Sperm Interaction Research Articles

Hybrid motility mechanism of sperm at viscoelastic fluid-solid interface

To fertilize eggs, sperm must pass through narrow, complex channels filled with viscoelastic fluids in the female reproductive tract. While it is known that the topography of the surfaces plays a role in guiding sperm movement, sperm have been thought of as swimmers, i.e., their motility comes solely from sperm interaction with the surrounding fluid, and therefore, the surfaces have no direct role in the motility mechanism itself. Here, we examined the role of solid surfaces in the movement of sperm in a highly viscoelastic medium. By visualizing the flagellum interaction with surfaces in a microfluidic device, we found that the flagellum stays close to the surface while the kinetic friction between the flagellum and the surface is in the direction of sperm movement, providing thrust. Additionally, the flow field generated by sperm suggests slippage between the viscoelastic fluid and the solid surface, deviating from the no-slip boundary typically used in standard fluid dynamics models. These observations point to hybrid motility mechanisms in sperm involving direct flagellum-surface interaction in addition to flagellum pushing the fluid. This finding signifies an evolutionary strategy of mammalian sperm crucial for their efficient migration through narrow, mucus-filled passages of the female reproductive tract.

The effect of mixed honeybee drone semen on sperm quality characteristics: A preliminary study

Following copulation, sperm of multiple honeybee (Apis mellifera (A.m.) Linnaeus (Apidae)) drones are mixed inside the spermatheca of a queen, and sperm behaviour in this environment is of importance for sperm survival and successful fertilization. The polyandrous mating system of honeybees further may allow for cryptic female choice and sperm competition to select for specific sperm traits to enable successful reproduction. This preliminary study therefore examined multiple sperm quality parameters in mixed drone semen. Thirty-one drone ejaculates were collected and split to be mixed with one and two other drones’ semen. A computerised sperm analysis system was used for analysing sperm functionality parameters, at baseline and 60 minutes. Morphology measurements included head, tail, and total sperm length. Sperm concentration was significantly higher in three-drone samples compared to other groups (p < 0.001), while comparisons of sperm functional and morphometric parameters did not reveal differences. However, after 60 minutes, mixed semen samples displayed changes in terms of sperm functionality. The motility percentage significantly decreased in two-drone samples (p = 0.004). In contrast, three-drone samples had consistently lower motility and kinematic parameters at baseline, but following an hour it was the only group to show improvement in sperm progressive motility, swimming speed and kinematics. Sperm concentration correlated with the majority of functionality parameters, while, morphological measurements did not. Sperm functionality changes observed in this study, can be attributed to a combination of factors, including, mixing of drone semen, sperm concentration, and time allowing for potential sperm interaction.

A longer period of epididymal sperm interaction with extender components during cryopreservation improves sperm quality, decreases the size of sperm distal cytoplasmic droplets, and changes the number of nanoparticles in the extender

While cryopreservation of cauda epididymal sperm (SpCau) allows the preservation of post-mortem bulls’ gametes, the process triggers sperm damage. Although improving post-thaw sperm quality, using egg yolk extenders (EY) raises biosafety concerns which forces the use of EY-free extenders (EYFE). Since EYFE are less efficient in preserving post-thaw sperm quality, a strategy for ejaculated sperm (SpEj) frozen with EYFE is to add an Equilibrium Time (ET) step period to the cryopreservation process. However, the ET effect on the quality of SpCau cryopreserved in EYFE remains unknown. Distinct from SpEJ, SpCau physiologically displays cytoplasmic droplets (CDs) in the flagellum that may benefit cell exchange during ET. We hypothesized that using ET in SpCau cryopreserved with EYFE impacts sperm morphofunctional features, CD area, and in vitro fertility ability. Extender nanoparticles were also assessed. Following collection from the cauda epididymis of six Nellore bulls by retrograde flow, SpCau were cryopreserved in EYFE BoviFree® (Minitube, Germany) using three ET protocols: ET0 (no-ET); ET2.5 (2.5 h-ET); and ET5 (5 h-ET). SpCau from ET2.5 and ET5 showed a higher (P ≤ 0.05) percentage of motility and integrity of plasma and acrosome membranes and a smaller (P ≤ 0.05) distal CD area. There are no differences in sperm abnormalities, oxidative stress, capacitation-like events, and in vitro fertility ability. However, a better sperm recovery was found after Percoll® selection for ET2.5 and ET5. Interestingly, the number of nanoparticles in the extender decreased in post-thawed samples. In conclusion, an ET of 2.5 or 5 h is required for an efficient SpCau cryopreservation using an EYFE.

Exosomes from uterine fluid promote capacitation of human sperm.

Extracellular vesicles (EVs) are membrane-bound vesicles containing various proteins, lipids, and nucleic acids. EVs are found in many body fluids, such as blood and urine. The release of EVs can facilitate intercellular communication through fusion with the plasma membrane or endocytosis into the recipient cell or through internalization of the contents. Recent studies have reported that EVs isolated from human endometrial epithelial cells (EECs) promote sperm fertilization ability. EVs from uterine flushing fluid more closely resemble the physiological condition of the uterus. However, it is unclear whether EVs derived directly from uterine flushing fluid have the same effect on sperm. This study aimed to research the effect of EVs from uterine flushing fluid on sperm. EVs were isolated from the uterine flushing fluid. The presence of EVs was confirmed by nanoparticle tracking analysis (NTA), Western blot, and transmission electron microscopy (TEM). EVs were incubated with human sperm for 2 h and 4 h. The effects of EVs on sperm were evaluated by analyzing acrosome reaction, sperm motility, and reactive oxygen species (ROS). The EVs fractions isolated from the uterine fluid were observed in cup-shaped vesicles of different sizes by TEM. All isolated vesicles contained similar numbers of vesicles in the expected size range (30-200 nm) by NTA. CD9 and CD63 were detected in EVs by western blot. Comparing the motility of the two groups incubated sperm motility significantly differed at 4 h. The acrosome reactions were promoted by incubating with EVs significantly. ROS were increased in sperm incubated with EVs. Our results showed EVs present in the uterine fluid. Acrosome reactions and ROS levels increased in human sperm incubated with EVs. EVs from uterine fluid can promote the capacitation of human sperm. The increased capacitation after sperm interaction with EVs suggests a possible physiological effect during the transit of the uterus.

Comparative effectiveness of cryoprotective diluents for rooster seed

The preservation and use of frozen/ thawed male sperm is important for maintaining the biodiversity of poultry and will eventually play an important role in breeding, allowing greater flexibility in breeding programs. This use of thawed semen is advisable only at a high level of its fertilizing ability. Trehalose is a promising natural component in cryoprotective media for rooster semen due to its exceptional ability to neutralize cold stress. The purpose of the study was to determine the effectiveness of using trehalose as part of a medium for cryopreservation of rooster semen based on the Russian development - Leningrad cryoprotective medium (LCM-control). The composition of the LCM-T20 experimental medium with the addition of trehalose at a concentration of 9.5 mM has been developed. The mobility indicators of thawed rooster semen (Rhode Island Red breed, ♂n=10) did not differ significantly depending on the composition of the medium. Significant differences (p <0.05) were obtained in terms of egg fertilization: 86.0% when using the LCM-T20 medium and 79.0% using LCMcontrol and in assessing the state of the vitelline membrane of the egg yolk (p <0.001). The fertilization rate of eggs at a level acceptable for thawed semen of 50% was maintained until the 10th day (20th day of egg collection) from the last insemination when using the LKS-T20 medium. The functional usefulness of thawed sperm was maintained for 15 days (the 25th day of egg collection) from the last insemination when using the LCM-T20 media - egg fertilization was 15.0%, the average number of points of interaction of sperm with the vitelline membrane of the egg yolk was 345.2 pcs/cm2. When using the LCM-control diluent, the functional ability of thawed spermatozoa was absent during the same control periods. The achieved high level of egg fertilization when trehalose was included in the cryoprotective diluent in combination with the method of freezing rooster semen in pellets confirms the feasibility of introducing the LCMT20 diluent into the technology for preserving the genetic diversity of local and indigenous breeds of chickens using in vitro methods.

Molecules involved in the sperm interaction in the human uterine tube: a histochemical and immunohistochemical approach

In humans, even where millions of spermatozoa are deposited upon ejaculation in the vagina, only a few thousand enter the uterine tube (UT). Sperm transiently adhere to the epithelial cells lining the isthmus reservoir, and this interaction is essential in coordinating the availability of functional spermatozoa for fertilization. The binding of spermatozoa to the UT epithelium (mucosa) occurs due to interactions between cell-adhesion molecules on the cell surfaces of both the sperm and the epithelial cell. However, in humans, there is little information about the molecules involved. The aim of this study was to perform a histological characterization of the UT focused on determining the tissue distribution and deposition of some molecules associated with cell adhesion (F-spondin, galectin-9, osteopontin, integrin αV/β3) and UT's contractile activity (TNFα-R1, TNFα-R2) in the follicular and luteal phases. Our results showed the presence of galectin-9, F-spondin, osteopontin, integrin αV/β3, TNFα-R1, and TNFα-R2 in the epithelial cells in ampullar and isthmic segments during the menstrual cycle. Our results suggest that these molecules could form part of the sperm-UT interactions. Future studies will shed light on the specific role of each of the identified molecules.

Correlation of the zinc level in the spermoplasm with the fertility characteristics of human ejaculate

Background: Zinc is essential for the normal functioning of the male reproductive system. The data on the diagnostic value of the zinc level in the human spermoplasm and its relationship with the main parameters of the sperm fertility are contradictory. Aim: study of the correlations between the zinc level in the spermoplasm and the spermogram characteristics. Methods: The sperm of men of the reproductive age (n=486, average age 33.07±3.03 years) was studied. In addition to the standard spermogram, MAR tests (IgA, IgG and IgM) were performed in the sperm samples, the degree of fragmentation of the sperm DNA, the sperm interaction with hyaluronic acid, the acrosine activity, and the neutral alpha-glucosidase activity were assessed, the citric acid, fructose and glycodelin levels were determined, and the level of reactive oxygen species was studied. The zinc level determination in the spermoplasm was carried out by a standard spectrophotometric method with 5-Br-PAPS chromogen. The Pearson's formula was used for the correlation analysis. The study was conducted from 2018 to May 2022, once. Results: A significant negative correlation of the zinc level in the spermoplasm with the age of men was revealed (r=-0.16; p 0.001). The level of zinc in the spermoplasm weakly negatively correlated with the dilution time and with the viscosity of the sperm. The positive correlation was found with the number of spermatozoa (r=0.13; p 0.01) and their mobility (r=0.38; p 0.00001). The level of zinc in the spermoplasm negatively correlated with the degree of the sperm DNA fragmentation and with the amount of reactive oxygen species, and positively correlated with the results of the test for binding to hyaluronic acid. Conclusions: The level of zinc in the spermoplasm significantly correlates with a number of physiological and biochemical characteristics of the sperm. The data obtained allow us to recommend determination of the zinc level in the sperm plasma to not only assess the functional activity of the prostate gland, but also to diagnose the fertility of the ejaculate, as well as to optimize the therapy with zinc-containing drugs and improve the control over the effectiveness of the treatment.

Sperm interaction with bacteria induces the spontaneous acrosome reaction

Bacterial contamination in the semen deteriorates spermatozoa function. One mechanism through which this may occur is by inducing a premature form of the acrosome reaction (spontaneous acrosome reaction (sAR)) which has been shown to abrogate fertilization. To understand the mechanism by which bacteria affect sperm functions, we determined the effects of bacteria on sperm sAR and on other parameters involved in sperm capacitation. Sperm cells undergo biochemical changes in the female reproductive tract collectively called capacitation. Only capacitated sperm can undergo the physiological acrosomal exocytosis process near or on the oocyte, which allows the spermatozoon to penetrate and fertilize the egg. Bovine sperm incubated with the bacteria Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) or Pseudomonas aeruginosa (P. aeruginosa), revealed a sperm-bacteria interaction, however only E. coli and P. aeruginosa caused an increase in sperm sAR. This effect was seen only when the bacteria were present with the sperm during the full incubation under capacitation conditions but not when the bacteria were added to capacitated sperm. These results indicate that bacteria affect sperm during capacitation and not at the AR step. In addition, Ca2+ influx, protein kinase A, and protein tyrosine phosphorylation activities, three essential processes that promote capacitation, were inhibited by the bacteria. Moreover, increasing intracellular cAMP, which also occur during sperm capacitation, caused significant reverse of sAR induced by the bacteria.

Cervical Abnormalities are related to Infertility: A Review

Infertility is a disease and cervical abnormalities of the genital tract in females may cause this condition. To evaluate the cervical abnormalities related to infertility in females a systematic review was done. Related literature were collected via Google Scholar, NCBI, PubMed, and Medscape. For article searching following keywords were used: cervical abnormalities, infertility, uterine anomalies, and cervical factors. As the literature reviewed, more than 60 articles were studied, from which only 45 articles were added. The study suggested that many cervical abnormalities affect fertility including the poor interaction of mucus and sperm in the cervix, cervical cancer, dilatation and stenosis of the cervix, wall irregularities, diverticulum, masses, and metaplasia of the cervix. Some of the microorganisms also affect cervical functions causing infertility. It is concluded that cervical abnormalities can be related to infertility.

TLR-1, TLR-2, and TLR-6 MYD88-dependent signaling pathway: A potential factor in the interaction of high-DNA fragmentation human sperm with fallopian tube epithelial cells.

The DNA integrity of spermatozoa that attach to fallopian tube (FT) cells is higher than spermatozoa that do not attach. FT epithelial cells can distinguish normal and abnormal sperm chromatin. This study investigated the effects of sperm with a high-DNA fragmentation index (DFI) from men with unexplained repeated implantation failure (RIF) on the Toll-like receptor (TLR) signaling pathway in human FT cells in vitro. Ten men with a RIF history and high-DFI and 10 healthy donors with low-DFI comprised the high-DFI (>30%) and control (<30%) groups, respectively. After fresh semen preparation, sperm were co-cultured with a human FT epithelial cell line (OE-E6/E7) for 24 hours. RNA was extracted from the cell line and the human innate and adaptive immune responses were tested using an RT2 profiler polymerase chain reaction (PCR) array. The PCR array data showed significantly higher TLR-1, TLR-2, TLR-3, TLR-6, interleukin 1α (IL-1α), IL-1β, IL-6, IL-12, interferon α (IFN-α), IFN-β, tumor necrosis factor α (TNF-α), CXCL8, GM-CSF, G-CSF, CD14, ELK1, IRAK1, IRAK2, IRAK4, IRF1, IRF3, LY96, MAP2K3, MAP2K4, MAP3K7, MAP4K4, MAPK8, MAPK8IP3, MYD88, NFKB1, NFKB2, REL, TIRAP, and TRAF6 expression in the high-DFI group than in the control group. These factors are all involved in the TLR-MyD88 signaling pathway. The MyD88-dependent pathway through TLR-1, TLR-2, and TLR-6 activation may be one of the main inflammatory pathways activated by high-DFI sperm from men with RIF. Following activation of this pathway, epithelial cells produce inflammatory cytokines, resulting in neutrophil infiltration, activation, phagocytosis, neutrophil extracellular trap formation, and apoptosis.

Insulin and Bull Sperm Interactions During Cryopreservation

Insulin and Bull Sperm Interactions During Cryopreservation

Signaling Proteins Recruited to the Sperm Binding Site: Role of β-Catenin and Rho A.

Sperm interaction with the oocyte plasma membrane triggers a localized response in the mouse oocyte that leads to remodeling of oocyte surface as well as the underlying cortical actin layer. The recent demonstration that PTK2B is recruited and activated at the sperm binding site raised the possibility that multiple signaling events may be activated during this stage of fertilization. The present study demonstrated that β-catenin and Rho A were recruited to the cortex underlying bound/fused sperm. To determine whether sperm-oocyte contact was sufficient to initiate β-catenin recruitment, Cd9-null, and PTK2b-null oocytes were tested for the ability to recruit β-catenin to sperm binding sites. Both Cd9 and Ptk2b ablation reduced β-catenin recruitment raising the possibility that PTK2B may act downstream of CD9 in the response to sperm binding/fusion. Further immunofluorescence study revealed that β-catenin co-localized with f-actin in the interstitial regions between actin layer fenestrae. Rho A, in contrast, was arranged underneath the actin layer in both the fenestra and the interstitial regions suggesting that they may play different roles in the oocyte.

Fine structure of micropylar regions of Cobitis hankugensis, Iksookimia longicorpa and their unisexual natural hybrids.

Iksookimia longicorpa and Cobitis hankugensis are two species of fish distributed only on the Korean Peninsula. They have a unique reproductive ecology that naturally hybridizes into three widely known unisexual types, maintaining populations of almost all females. In this study, the fine structure of the micropyles of I. longicorpa, C. hankugensis and their hybrids was analyzed to find out how egg-sperm interaction, a common interspecies isolation mechanism, is possible between heterogeneous species. Analysis of 30 eggs from five females of each species revealed that all had one funnel-shaped micropylar region and a manhole-shaped micropyle canal. With the exception of C. hankugensis, which had no spiral grooves or ridges, the rest had counterclockwise spiral grooves and ridges on the micropylar region. All five species, however, showed identical groove patterns for the micropyle canal. The egg size was the largest in HL (one from the C. hankugensis locus with one from the I. longicorpa locus) and the smallest in C. hankugensis. In the hybrids, the HL type had the largest egg and HHL (two from the C. hankugensis locus with one from the I. longicorpa locus) type the smallest. For the diameter of the micropylar region and micropyle canal, the diploid I. longicorpa, C. hankugensis and HL were smaller than those of the triploid. In addition, as the ratio of the canal diameter to the eggs was lower in I. longicorpa than in C. hankugensis, it was confirmed that I. longicorpa has a relatively small micropyle canal compared with C. hankugensis.

Cervix chip mimicking cervical microenvironment for quantifying sperm locomotion

As the gate for sperm swimming into the female reproductive tract, cervix is full of cervical mucus, which plays an important role in sperm locomotion. The fact that sperm cannot pass through the cervical mucus-cervix microenvironment will cause the male infertility. However, how the sperm swim across the cervix microenvironment remains elusive. We used hyaluronic acid (HA), a substitute of cervical mucus to mimic cervix microenvironment and designed a cervix chip to study sperm selection and behavior. An accumulation of sperm in HA confirmed that HA served as a reservoir for sperm, similar to cervical mucus. We found that sperm escaping from HA exhibited higher motility than the sperm accessing into HA, suggesting that HA functions as a filter to select sperm with high activity. Our findings construct a practical platform to explore the sophisticated interaction of sperm with cervix microenvironment, with elaborate swimming indicators thus provide a promising cervix chip for sperm selection with kinematic features on-demand. What's more, the cervix chip allows the convenient use in clinical infertility diagnosis, owing to the advantage of simple, fast and high efficiency.

Progesterone, Myo-Inositol, Dopamine and Prolactin Present in Follicular Fluid Have Differential Effects on Sperm Motility Subpopulations.

Considering the challenges surrounding causative factors in male infertility, rather than relying on standard semen analysis, the assessment of sperm subpopulations and functional characteristics essential for fertilization is paramount. Furthermore, the diagnostic value of sperm interactions with biological components in the female reproductive tract may improve our understanding of subfertility and provide applications in assisted reproductive techniques. We investigated the response of two sperm motility subpopulations (mimicking the functionality of potentially fertile and sub-fertile semen samples) to biological substances present in the female reproductive tract. Donor semen was separated via double density gradient centrifugation, isolated into high (HM) and low motile (LM) sperm subpopulations and incubated in human tubal fluid (HTF), capacitating HTF, HD-C medium, progesterone, myo-inositol, dopamine and prolactin. Treated subpopulations were evaluated for vitality, motility percentages and kinematic parameters, hyperactivation, positive reactive oxygen species (ROS), intact mitochondrial membrane potential (MMP) and acrosome reaction (AR). While all media had a significantly positive effect on the LM subpopulation, dopamine appeared to significantly improve both subpopulations’ functional characteristics. HD-C, progesterone and myo-inositol resulted in increased motility, kinematic and hyperactivation parameters, whereas prolactin and myo-inositol improved the LM subpopulations’ MMP intactness and reduced ROS. Furthermore, progesterone, myo-inositol and dopamine improved the HM subpopulations’ motility parameters and AR. Our results suggest that treatment of sub-fertile semen samples with biological substances present in follicular fluid might assist the development of new strategies for IVF treatment.

Sperm interaction with the uterine innate immune system: toll-like receptor 2 (TLR2) is a main sensor in cattle.

During the passage through the female reproductive tract, sperm interact with various compartments and their immune systems. The immune system that protects the female against pathogens also could destroy sperm or prevent them from reaching the site of fertilisation. In particular, the uterine innate immune response is crucial from the perspectives of both the sperm and the uterus. Following insemination, sperm immediately start to trigger inflammation in the uterus by entering uterine glands and activating an innate immune response. In cattle, the activation occurs mainly via TLR2 signalling, if not the only one, between sperm and the uterine epithelium lining the glands. This acute immune response is manifested as the upregulation of mRNA expression of IL8, TNFA, IL1B , and PGES . As a consequence, many sperm are trapped by polymorphonuclear neutrophils, the first and major component of innate immunity. The sperm-induced uterine innate immune responses apparently serve to clear the uterus of excess sperm and, importantly, prepare the endometrium for implantation. Pathophysiological conditions in the uterus seriously disrupt this phenomenon, and thus could directly decrease fertility.

The conserved fertility factor SPACA4/Bouncer has divergent modes of action in vertebrate fertilization

Fertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm-egg interaction is incomplete, partly because many of the essential factors found in nonmammalian species do not have obvious mammalian homologs. We have recently identified the lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) protein Bouncer as an essential fertilization factor in zebrafish [S. Herberg, K. R. Gert, A. Schleiffer, A. Pauli, Science 361, 1029-1033 (2018)]. Here, we show that Bouncer's homolog in mammals, Sperm Acrosome Associated 4 (SPACA4), is also required for efficient fertilization in mice. In contrast to fish, in which Bouncer is expressed specifically in the egg, SPACA4 is expressed exclusively in the sperm. Male knockout mice are severely subfertile, and sperm lacking SPACA4 fail to fertilize wild-type eggs in vitro. Interestingly, removal of the zona pellucida rescues the fertilization defect of Spaca4-deficient sperm in vitro, indicating that SPACA4 is not required for the interaction of sperm and the oolemma but rather of sperm and the zona pellucida. Our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization.

Does the Rainbow Trout Ovarian Fluid Promote the Spermatozoon on Its Way to the Egg?

The fertilization of freshwater fish occurs in an environment that may negatively affect the gametes; therefore, the specific mechanisms triggering the encounters of gametes would be highly expedient. The egg and ovarian fluid are likely the major sources of these triggers, which we confirmed here for rainbow trout (Oncorhynchus mykiss). The ovarian fluid affected significantly spermatozoa performance: it supported high velocity for a longer period and changed the motility pattern from tumbling in water to straightforward moving in the ovarian fluid. Rainbow trout ovarian fluid induced a trapping chemotaxis-like effect on activated male gametes, and this effect depended on the properties of the activating medium. The interaction of the spermatozoa with the attracting agents was accompanied by the “turn-and-run” behavior involving asymmetric flagellar beating and Ca2+ concentration bursts in the bent flagellum segment, which are characteristic of the chemotactic response. Ovarian fluid created the optimal environment for rainbow trout spermatozoa performance, and the individual peculiarities of the egg (ovarian fluid)–sperm interaction reflect the specific features of the spawning process in this species.

P–099 MYD88 dependent pathway through TLR 1,2 and 6 activation play a role in interaction of high DNA fragmented human sperm with fallopian tube epithelial cells

Abstract Study question How does sperm with high DFI disrupt the female reproductive system? Summary answer Sperm with damaged DNA could disrupt the female reproductive system by production of high inflammatory factors through activation of TLRs and MyD88-dependent pathway . What is known already Previous studies have also shown that the interaction between sperm and fallopian tube is a complex process and activates a number of immune mechanisms. Many TLRs, including TLR1, TLR2, and TLR6 are expressed in fallopian tube epithelial cells. TLRs have an important role in the immune interaction between fallopian tube and sperm. They are also involved in sperm capacitation, fertilization and pregnancy. Examining this interaction may lead us to valuable data. Study design, size, duration In this study, 10 Recurrent Implantation failure couples with high DFI ( &amp;gt;30% by SCSA evaluation) and 10 healthy donors men with low DFI (&amp;lt;30%) were considered as high DFI group and low DFI group respectively. After fresh semen preparation, sperm were co-cultured with human fallopian tube epithelial cell line( OE-E6/E7) for 24 hours. Participants/materials, setting, methods RNA were extracted from the cell line and PCR array for human innate and adaptive immune responses was performed by RT2 profiler PCR array. Main results and the role of chance Analysis of PCR array data showed that the expression of TLR–1, TLR–2, TLR–6, MYD88, TIRAP, IRAKS,TRAF6, MAPKS, NF-KB, G-CSF, GM-CSF, CXCL8, CXCL10, CCL2, IL–6, IL–1, TNFa in high DFI group was significantly higher than control group. All these factors are involved in the MyD88-dependent pathway. Our research suggests that the MyD88-dependent pathway through TLR1,2 and 6 activation may be the main inflammatory pathway activated by sperm with high DFI. The released DAMPs through damaged sperms were recognized by TLRs on the epithelial cells. Following the TLR1,2 and 6 signaling, and MYD88 activation, epithelial cells were producing the inflammatory cytokines which result in the neutrophils infiltration. In the lumen of the fallopian tube, the neutrophils were activated via TLR1/TLR2, complexes that lead to the neutrophils activation, phagocytosis, NET formation, and apoptosis. Limitations, reasons for caution Studying with a larger sample size and examining the final factors at the protein level will be better to detect the effect of sperm DNA fragmentation on fallopian tube. Wider implications of the findings: MyD88-dependent pathway could be one of the mechanisms that involved in interaction of high DNA fragmented sperm with female reproductive tract. Trial registration number Not applicable

Synergistic interactions of sperm impairing bacteria: Impact on pregnancy outcome in mouse model

Earlier in our laboratory, the role of various individual sperm impairing microorganisms on sperm parameters and female infertility has been elucidated at higher doses. As, multiple bacterial species tend to exert more pathogenic effect in comparison to single organism hence, present study was carried out to evaluate that if consortia of these sperm impairing organisms can lead to infertility in female mice at sub fertility dose. For this, impact of individual bacterial strains of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Klebsiella pneumoniae and consortia of Escherichia coli and Pseudomonas aeruginosa, Escherichia coli and Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae was examined on the motility, viability of mouse spermatozoa and fertility outcome. The results showed that the individual bacterial strains of E. coli, S. marcescens and K. pneumoniae could led to immobilization of spermatozoa by agglutination and P. aeruginosa led to immobilization of spermatozoa without agglutination. Also, all of them led to 100% sperm death in 45 min of incubation. In case of consortia of bacterial strains, the results showed sperm agglutination in all the cases and they were able to induce 100% sperm death at 30 min of incubation time. Further, in vivo studies were carried out to evaluate the impact of individual bacterial strains and consortia of bacterial strains on the fertility outcome in female Balb/c mice. For this, female mice were administered intravaginally with 101 cfu/20µl of individual bacterial strains or consortia of strains for 10 consecutive days or PBS. The results showed that both individual bacterial strains and consortia of bacterial strains were able to efficiently colonize the mouse vagina. Further, control group receiving phosphate buffer saline and groups receiving individual bacterial strains showed all the pregnancy related changes viz. abdominal distension, string of pearls on palpation as well as delivery of pups on completion of gestation period and delivery of pups. The histological examination of reproductive organs viz. uterus and ovary, of the female mice receiving PBS or individual bacterial strains showed the formation of corpus luteum in the ovary and the formation of decidua’s in the uterus, indicative of pregnancy. However, mice receiving consortia of bacterial strains did not show any pregnancy related changes throughout the experiment. Thus, these results indicate that the presence of consortia of sperm impairing microorganisms in vaginal milieu is efficient in provoking infertility even at subfertility doses.