Sperm Immobilization Research Articles

Automated Piezo-Assisted Sperm Immobilization

Sperm immobilization is a crucial procedure in clinical cell surgery for infertility treatment. Current immobilization is implemented by tapping the sperm tail with a glass micropipette, but its effectiveness is restricted by sperm orientation and ineffective membrane ablation. Ineffective ablation leads to limited release of oocyte activating factors and lowers fertilization rate; and sperm swim in small angles relative to the micropipette tip cannot be tapped due to the risk of damaging the sperm’s genetic materials contained in the sperm head. This paper reports automated piezo-assisted sperm immobilization with enhanced efficacy of cell membrane ablation and sperm orientation control. The designed piezo drill consists of two orthogonal vibration modules to generate controlled micropipette vibration along axial and lateral axes. Through stiffness modeling, the flexure joints guide the motion of the central beam of each vibration module. To achieve sperm orientation control, whirl flow is induced by both axial and lateral vibration of the micropipette tip. To immobilize sperm, only micropipette’s axial vibration is generated to prevent lateral vibration from damaging sperm head. A visual servoing scheme is developed by decoupling sperm wiggling from positioning error for immobilization. Experimental results showed that sperm orientation control by the piezo drill achieved an error of 1.4 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$^{\circ}$</tex-math> </inline-formula> and a time cost of 2.5 s. Visual servoing with sperm wiggling decoupling achieved a positioning error of 1.7 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\mu$</tex-math> </inline-formula> m. Furthermore, the piezo-assisted sperm immobilization technique led to effective membrane ablation. With membrane-impermeable stains, it took 5.6 s for the immobilized sperm to be stained after piezo-assisted immobilization, significantly less than 49.2 s by conventional micropipette tapping. <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Note to Practitioners</i> —This work tackled the challenge of ineffective membrane ablation and orientation limit in clinical cell surgeries. Conventional manual immobilization suffers from low membrane ablation efficacy, which leads to limited release of oocyte activating factors and lowers fertilization rate. Moreover, sperm swim in small angles relative to the micropipette tip cannot be tapped due to the risk of damaging the sperm’s genetic materials contained in the sperm head. In this paper, we propose automation techniques for effective membrane ablation and orientation control of sperm. A clinically compatible piezo drill is developed to generate controllable micropipette motion along both axial and lateral directions. The whirl flow generated by micropipette vibration is employed to rotate sperm, which greatly increased the number of available sperm for immobilization. A visual servoing controller is developed to keep the sperm at the center of field of view for immobilization by decoupling sperm wiggling from positioning error. The developed methods can be generalized to the manipulation of other types of cells. The piezo drill can be used for effective membrane ablation of oocyte, embryo, yeast cell and so on. The orientation control strategy leveraging piezo-induced whirl flow is applicable to non-contact rotation of a variety of microorganism.

Advancements in the detection and implications of sperm-immobilizing antibodies in female infertility

This review highlights over five decades of research on sperm-immobilizing antibodies (SI-Abs), which are crucial for understanding female infertility due to their effects on sperm motility and fertilization. Since the 1960s, Isojima et al. have made significant strides, notably with the Sperm Immobilization Test (SIT), which revolutionized the quantification of SI-Abs and their roles in infertility. Drawing from a comprehensive PubMed search on "the sperm immobilization test" and "sperm immobilizing antibody," our review underscores the critical insights gained into SI-Abs' impact on reproductive functions. SI-Abs result from the body's response to sperm antigens, potentially leading to infertility by affecting post-intercourse sperm function. However, the presence of anti-sperm antibodies does not guarantee infertility, indicating a complex relationship between these antibodies and reproductive outcomes. Isojima et al.'s pioneering studies paved the way for SIT and sperm immobilization titer (SI50), tools that have clarified the link between SI-Abs and infertility, focusing on disrupted sperm mobility and fertilization as key infertility mechanisms. Clinically, interventions such as in-vitro fertilization (IVF), which bypasses or eliminates SI-Abs, have improved pregnancy rates, whereas Freund's complete adjuvant therapy has deepened our understanding of infertility mechanisms. The SI50 value is crucial for predicting fertility treatment success and guiding therapeutic decisions based on antibody levels. In summary, the evolution of SI-Abs research has provided new hope for addressing infertility, significantly enriching the field of reproductive immunology, and highlighting the need for ongoing investigation.

A Perspective on Male Contraceptive Therapeutics of Hydro-ethanol Extract of Seeds of &lt;i&gt;Luffa acutangula (L.) Roxb.&lt;/i&gt; in Human and Albino Rat: An &lt;i&gt;In-vitro&lt;/i&gt; Meta-Analysis Study

Traditionally Luffa acutangula (L.) Roxb. has been utilized as an herbal contraceptive. This in-vitro study has been performed to search out the male contraceptive efficacy of hydro-ethanol (60:40) extract of Luffa acutangula (LAHEE) seeds in a dose-specific manner (1, 2, and 4 mg/ml of in-vitro media). The percentages of motile, viable, hypo-osmotic swelled (HOS), and acrosomal intact of human and rat sperms were declined significantly (p&lt;0.05) at the above-mentioned doses of LAHEE-exposed groups against the placebo group. The inhibitory concentration 50% value (IC50) of LAHEE was 2.5 mg/ml in human and 1.2 mg/ml in rat spermatozoa immobilization. The ∆5,3β and 17β hydroxysteroid dehydrogenase (HSD) activities of rat’s testicular tissues were inhibited significantly at p&lt;0.05 in LAHEE-treated groups than the placebo group. Activities of superoxide dismutase (SOD) and catalase were significantly inhibited (p&lt;0.05) along with the significant increment (p&lt;0.05) in the quantity of thiobarbituric acid reactive substances (TBARS) in rat’s testes and epididymis, sperm pellets of humans and rats in LAHEE-treated groups against the placebo group without any significant difference (p&gt;0.05) in above said sensors in the liver and cardiac tissues of rats. The non-toxic nature of LAHEE was indicated by no significant alterations (p&gt;0.05) in the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) after LAHEE exposure in said tissues of rats. The LC-MS study of LAHEE identified the presence of oleic acid, dihydroquercetin, luteolin-7-glucoside, formononetin, luteolin 8-C-pentoside-6-C-hexoside, pterosin B, boldine and berberine. These findings interpreted that the LAHEE possesses spermiological inhibitory and hypo-testicular activities, which indicate a promising possibility to develop a potent herbal male contraceptive agent from this plant extract.

Aggressive sperm immobilization improves reproductive outcomes in patients with suboptimal semen parameters and previous ICSI fertilization failure

Intracytoplasmic sperm injection (ICSI) is the most effective procedure to resolve male infertility, enhancing overall fertilization and pregnancy outcomes. However, it is important to note that fertilization failure (FF) can still occur in a few cases after ICSI. This study aims to introduce a specialized technique of aggressive sperm immobilization for ICSI and evaluate its impact on reproductive outcomes in cases involving prior fertilization failure. All infertile couples with male partners having suboptimal semen samples and previous ICSI fertilization failure were evaluated using retrospective data from National Taiwan tertiary university hospital (NTUH) between January 2016 and February 2022. Fertilization failure in our study was defined as less than 30% fertilization rate (FR, the number of normally fertilized oocytes divided by the total number of injected mature oocytes). Data involving both standard (routine procedure) and aggressive sperm immobilization (SI) techniques during different ICSI cycles were included in this study. Standard and aggressive SI methods were performed by compressing the distal half tail of the spermatozoa ≦ 5 and 15 times prior to ICSI respectively. Generalized estimating equations analysis were applied to compare the clinical outcomes between two procedures. Overall, data from 23 infertile couples who had undergone 65 ICSI cycles (31 standard SI with low fertilization rate and 34 aggressive SI) were included in the study. The average FR in the ICSI cycles with standard SI and aggressive SI were 23.6 ± 23.1% and 49.5 ± 31.8 respectively (P = 0.0002). The majority of embryos were transferred at the day 3 stage, with an average number transferred of 2.6 ± 0.9 in the aggressive SI group and 1.9 ± 0.9 in the standard group. The number of embryos transferred per transfer cycle was higher in the aggressive SI (P = 0.015), whereas the number of good-quality embryos was similar between the two procedures (P = 0.44). There were one and seven live births from the standard SI cycles and aggressive SI cycles respectively. In conclusion, aggressive SI was associated with a significantly higher FR, resulting in more available embryos for transfer without compromising embryo quality. Therefore, this specialized technique improved pregnancy outcome among infertile couples with a previous ICSI–FF. It can be a safe, economic, and effective method to improve the assisted reproductive technologies outcomes for infertile patients affected by previous ICSI–FF.

An experimental study to decipher the implications of antigenic sharing between Proteus mirabilis and mouse spermatozoa in eliciting an antisperm immune response: A potential culprit in immune infertility

The present study aims to investigate the antigenic cross reactivity between the receptor from Proteus mirabilis and spermatozoa against a common sperm immobilization factor, SIF, by calorimetric and competitive inhibition studies, and the immunogenicity of this receptor to evoke the formation of antisperm antibodies and their subsequent role in fertility outcome. The sperm binding receptor from Proteus mirabilis (PM-SBR) was extracted from ultrasonicated cell debris by treating it for 12 h at 37°C with 1 M NaCl. After being purified by gel permeation chromatography, its molecular weight as determined by SDS-PAGE was observed to be ≈ 47 kDa. The detrimental impacts of Sperm immobilizing factor (SIF) on spermatozoa viz. motility, viability, and morphology were mitigated when SIF was preincubated with various concentrations of PM-SBR. Using isothermal titration calorimetry, the entropy of the SIF-PM-SBR interaction was found to be -18.31 kJ/mol, whereas the free energy was 28.4 J/mol K. FTIR analysis was used to evaluate the binding interactions between PM-SBR and SIF. In addition, mice that were administered antibodies against PM-SBR were unable to conceive, in contrast to mice that were administered Phosphate buffer saline (PBS) or pre-immunization serum as controls. In light of this, we may conclude that anti-PM-SBR antibodies act as anti-sperm antibodies. Our work found that molecular mimicry between Proteus mirabilis and spermatozoa may cause antisperm immune reactivity. As a result of an immunological response to PM-SBR, infected individuals may produce antibodies against an epitope similar to one found on spermatozoa which helps in developing new strategies for managing autoimmune responses and infertility.

Bovine reproductive immunoinfertility: pathogenesis and immunotherapy.

Infertility is one of the primary factors for cattle reproduction in the present scenario. Reproduction-related immunoinfertility mainly involves immunization against the antigens related to reproductive hormones (LHRH, GnRH, Gonadal steroids, PGF2α and oxytocin), spermatozoa, seminal plasma and ovum. Anovulation, delayed ovulation, sperm immobilization, failure of fertilization, prolonged uterine involution, extended calving interval, prolonged post-partum estrus and reduced conception rate could be a result of immunoinfertility that occur due to the blockage of receptor site by antibodies formed against hormones, sperm and ovum. Immunoinfertility can be treated in the animal by giving sexual rest to females, by using various reproductive technologies such as in-vitro fertilization, gamete intra fallopian tube transfer, and intracytoplasmic sperm injection, sperm washing and by treating the animals with immunomodulators such as LPS, Oyster glycogen, etc. This review summarizes the different causes of bovine reproductive immunoinfertility and amelioration strategies to overcome it.

LALAPG variant of the Human Contraception Antibody (HCA) reduces Fc-mediated effector functions while maintaining sperm agglutination activity.

High rates of unintended pregnancies worldwide indicate a need for more accessible and acceptable methods of contraception. We have developed a monoclonal antibody, the Human Contraception Antibody (HCA), for use by women in vaginal films and rings for contraception. The divalent F(ab')2 region of HCA binds to an abundant male reproductive tract-specific antigen, CD52g, and potently agglutinates sperm. Certain other antibody activities mediated by the Fc region such as mucus trapping, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) could have beneficial or negative effects. The purpose of this study was to document HCA Fc effector functions and determine whether an engineered variant of HCA with a modified Fc region, HCA-LALAPG, retains desirable contraceptive activity while minimizing Fc-mediated effects. Fab and Fc functions were compared between HCA and HCA-LALAPG. Fab activity was assessed using sperm agglutination and modified swim-up ("sperm escape") assays. Fc functions were assessed by CDC (sperm immobilization), ADCP, and cervical mucus penetration assays. HCA and HCA-LALAPG showed equivalent activity in assays of Fab function. In the assays of Fc function, HCA supported strong CDC, ADCP, and sperm trapping in cervical mucus whereas HCA-LALAPG demonstrated little to no activity. HCA and the HCA-LALAPG variant were both highly effective in the sperm agglutination assays but differed in Fc mediated functions. Use of the HCA-LALAPG variant for contraception in women could reduce antibody-mediated inflammation and antigen presentation but may have reduced contraceptive efficacy due to much weaker sperm trapping in mucus and complement-dependent sperm immobilization activity.

Characterization of a spontaneously occurring self-reactive antibody against sperm in mice

We previously established a spontaneously occurring monoclonal antibody, namely Ts3, that was reactive to sperm from an aged male mouse. The present study investigated the characteristic properties and reproductive functions of Ts3. Immunofluorescent staining revealed that Ts3 reacted to epididymal sperm, and the corresponding antigen was located in the midpiece and principal piece. Immunohistochemistry revealed positive reactions in the germ cells and Sertoli cells in the testis, the epithelial cells in the epididymis and vas deferens. Through western blotting with two-dimensional electrophoresis, we demonstrated that Ts3 reacted with four spots, which were around Mr ∼25,000–60,000 and pI 5–6. MALDI-TOF/TOF mass spectrometry identified outer dense fiber 2 (ODF2) as a candidate for Ts3. ODF2 is a cytoskeletal structural component located in the midpiece and principal piece of the flagella of mammalian sperm. This was validated with the result of immunofluorescent staining, suggesting that ODF2 was the main target antigen for Ts3. Sperm immobilization test showed that Ts3 possessed sperm immobilizing activity. Furthermore, Ts3 impaired early embryo development but not in vitro fertilization. These results suggest that ODF2 plays an important role in both sperm function and early embryonic development.

Near-anoxia induces immobilization and sustains viability of sperm stored in ant queens

After copulation, insect females store sperm in a spermatheca for some duration until fertilization. At the beginning of their adult lives, ant queens can preserve numerous viable sperm cells from copulation for over ten years. However, the key factors influencing long-term sperm storage have not been identified. Here we show that the spermathecal environment is nearly anoxic, which induces sperm immobilization. Furthermore, mitochondrial respiratory inhibitors suppress sperm motility, suggesting that sperm immobilization may be caused by a shortage of ATP generated from only glycolysis under near-anoxic conditions. Sperm immobilization is not induced by acidification via glycolytic metabolism because the spermathecal fluid is not acidic. Finally, we show that artificial anoxic conditions rather than aerobic conditions sustain viable sperm cells. Therefore, near-anoxia is a key factor influencing long-term sperm storage in ant queens. The viability of sperm cells under artificial anoxia, however, is lower than that of those dissected immediately from queens. Moreover, the immotile sperm cells under more than 4 h of anoxia do not begin swimming after aerobic exposure, unlike those under anoxic conditions for less than 2 h. This finding indicates that factors other than anoxia are also necessary for long-term sperm preservation.

Role of the Glycogen Synthase Kinase 3-Cyclic AMP/Protein Kinase A in the Immobilization of Human Sperm by Tideglusib.

Tideglusib is considered to be a promising alternative to nonyl alcohol-9 contraceptives. Previous studies have demonstrated that the rapid spermicidal effect of tideglusib at a high concentration (≥ 10μM) may occur through detergent-like activity; however, the effect of low concentrations of tideglusib (< 5μM) on sperm is unknown. We explored the intracellular mechanism of tideglusib (< 5μM) on the immobilization of human sperm by exploring related signaling pathways in human sperm. After treatment with tideglusib (1.25μM) for 2h, sperm motility rate decreased to 0, while sperm membrane integrity rate was 70%. Protein tyrosine phosphorylation level and intracellular cyclic adenosine 3,5-monophosphate (cAMP) concentration decreased significantly compared to those in the control group. Isobutylmethylxanthine and 8-Bromo-cAMP relieved the inhibition of spermatozoa tyrosine phosphorylation, while tyrosine phosphorylation of sperm protein in the H89 and CALP1 treatment groups was significantly inhibited, and there was no difference in the tideglusib treatment group. H-89 and CALP1 reduced the level of serine phosphorylation of GSK-3α/β (Ser21/9), while its level was enhanced by IBMX and 8-Bromo-cAMP. Our results show the existence of the GSK3-cAMP/PKA regulatory loop in human sperm, which may mediate the immobilization effect of tideglusib at low of concentrations (e.g., 1.25μM) on sperm motility.

Automated End-Effector Alignment in Robotic Micromanipulation

Proper alignment of the end-effector is a critical procedure that determines the success of micromanipulation, such as robotic cell manipulation. Presently, end-effector alignment is performed manually and suffers from large misalignment error and inconsistency. Manual alignment often undesirably moves the end-effector (e.g., a glass micropipette) out of the limited field of view under microscopy and risks breaking the fragile end-effector. This article presents automated end-effector alignment in robotic micromanipulation. A rotational degree of freedom was added to a standard micromanipulator with translational degrees of freedom. The kinematic model of end-effector’s rotation was established, and the unknown model parameters were calibrated. To accommodate model uncertainty and parameter variations, a sliding mode controller was designed to achieve end-effector alignment. Experimental results demonstrate that the robotic alignment technique achieved an accuracy of 0.5 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$\pm 0.3^{\circ }$</tex-math></inline-formula> and a time cost of 17.9 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$\pm$</tex-math></inline-formula> 7.3 s, both significantly less than manual alignment. The developed controller based on kinematic modeling and sliding mode control achieved a higher success rate and significantly less time cost for end-effector alignment than the PID controller. Standard micropipettes were used as the end-effectors for sperm immobilization and oocyte penetration, important procedures in cell surgeries. The success rate of sperm immobilization was 98% by robotic micropipette alignment, higher than the success rate of 90% by manual alignment. Oocyte deformation before penetration was 28.1 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$\pm$</tex-math></inline-formula> 7.5 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$\mu$</tex-math></inline-formula> m by robotic end-effector alignment, significantly less than the deformation of 54.5 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$\pm$</tex-math></inline-formula> 13.2 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$\mu$</tex-math></inline-formula> m by manual alignment.

Bovine ICSI: limiting factors, strategies to improve its efficiency and alternative approaches

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique mainly used to overcome severe infertility problems associated with the male factor, but in cattle its efficiency is far from optimal. Artificial activation treatments combining ionomycin (Io) with 6-dimethylaminopurine after piezo-ICSI or anisomycin after conventional ICSI have recently increased the blastocyst rate obtained. Compounds to capacitate bovine spermatozoa, such as heparin and methyl-β-cyclodextrin and compounds to destabilize sperm membranes such as NaOH, lysolecithin and Triton X-100, have been assessed, although they have failed to substantially improve post-ICSI embryonic development. Disulfide bond reducing agents, such as dithiothreitol (DTT), dithiobutylamine and reduced glutathione, have been assessed to decondense the hypercondensed head of bovine spermatozoa, the two latter being more efficient than DTT and less harmful. Although piezo-directed ICSI without external activation has generated high fertilization rates and modest rates of early embryo development, other studies have required exogenous activation to improve the results. This manuscript thoroughly reviews the different strategies used in bovine ICSI to improve its efficiency and proposes some alternative approaches, such as the use of extracellular vesicles (EVs) as 'biological methods of oocyte activation' or the incorporation of EVs in the in vitro maturation and/or culture medium as antioxidant defence agents to improve the competence of the ooplasm, as well as a preincubation of the spermatozoa in estrous oviductal fluid to induce physiological capacitation and acrosome reaction before ICSI, and the use of hyaluronate in the sperm immobilization medium.

Symposium 1-4 - Immunological infertility caused by gamete immunity

1. Infertility caused by anti-sperm antibodies (ASA) 1) Female infertility caused by sperm-immobilizing antibodies in the sera In some women, ASA are produced as allogenic immunity, which causes infertility. Various methods for the antibody detection have been developed, but sperm-immobilization antibodies (SI-Ab) detected by the sperm immobilization test (SIT) have the greatest influence on the development of infertility. Sperm-immobilizing antibodies are present in the female genital tract, and also in the follicle fluid.SI-Ab cause a post-coital test (PCT) negative or impaired sperm transport from the uterine cavity to the Fallopian tubes, and fertilization disorders. There is a method to quantitatively measure SI-Ab titer, SI50 unit, and treatment methods are decided based on this SI 50 value. In cases with a SI50 value of 10 or more, it is difficult to conceive with AIH, and IVF is effective after preventing antibody contamination into the culture medium. As a mechanism for producing sperm-immobilizing antibodies, it has been speculated that women with disease-specific genes may be exposed to enough amount of sperm through sexual intercourse and develop symptoms triggered by such as chlamydia infection. CD52 is considered to be a promising antigen against sperm-immobilization antibodies, and is expected to be applied for the development of contraceptive vaccines. 2) Male infertility caused by ASA that bind to ejaculated sperm In some men, ASA are produced as autoantibodies, causing infertility. In men, it is not useful for measuring serum antibodies, therefore antibodies that bind to ejaculated sperm (sperm-binding antibodies) should be detected with ImmunoSpheres○R. However, since there are antibodies that are not related to the development of infertility, the treatment strategy can be determined by confirming whether the antibody has a biological effect. Inhibitory effect on cervical sperm penetration is confirmed by PCT and fertilization disorder is examined by the hemizona assay (HZA). If HZA cannot be performed, ICSI is recommended because it is likely to be accompanied by fertilization disorder in cases where the ratio of ASA-bound sperm to total motile sperm is 80% or more. 2. Female infertility caused by anti-zona pellucida (ZP) antibodies The ZP plays an important role in terms of egg formation, fertilization, protection of the embryo. These include the recognition of sperm species of animals, the induction of acrosome reaction, and the prevention of polysperm fertilization. On the other hand, since there is a strong antibody production ability as an antigen, if an anti-ZP antibodies are produced, the function of the ZP is impaired by the antibody, and there is a possibility of developing infertility. It was reported that anti-ZP antibodies were detected at a high rate in the sera of infertile women of unknown cause by the anti-ZP antibody detection method using porcine ZP, followed by controversial reports by another investigators. Therefore, we developed a detection method using human ZP. Anti-ZP antibodies disturb the follicular development, inhibit sperm-ZP binding and hatching. For the purpose of appropriate treatments, it was shown that ICSI is effective for fertilization disorder and assisted hatching (AHA) is effective for hatching disorder, respectively.

OP-17 - Characterization of spontaneously-occurring self-reactive antibody to sperm and its effects on in-vitro fertilization

Anti-sperm antibodies detected in humans possibly cause infertility. The mechanism of antibody production and the corresponding antigen are still unknown. Previously, we established a monoclonal antibody (named Ts3) reactive to sperm from an aged male mouse without any immunization. This study aims to identify the target self-antigen of Ts3 and to examine its effects on in-vitro fertilization. To examine the localization of the Ts3 recognizing antigen, mouse epididymal sperm were collected for immunofluorescent staining. The sperm proteins were extracted for two-dimensional electrophoresis and western blot analysis. Positive spots were subjected to mass spectrometry (MS). To determine influence on sperm motility, sperm immobilization test (SIT) with Ts3 was carried out. Further, the effects of Ts3 on mouse in-vitro fertilization and the developmental capacity of preimplantation embryos were investigated. Ts3 reacted to the midpiece and principal piece of mouse sperm. The detected molecular weight of the antigen to Ts3 was around 37 kDa, and the molecule was identified as outer dense fiber protein 2 (ODF2) by MS. In SIT, Ts3 was suggested to have harmful effect on sperm motility. Ts3 inhibited embryo development but not fertilization in mouse. Immunofluorescent staining of mouse preimplantation embryo showed Ts3 reacted to the membrane of the embryo and the areas of cellular contact. This study showed that Ts3, a spontaneously-occurring self-reactive antibody to sperm, recognized ODF2 as a targeted antigen. Although Ts3 did not inhibit fertilization, it impaired the sperm motility and the development of preimplantation embryo. Therefore, ODF2 antigen may be a pathogenic factor in infertility.

Robotic Rotational Positioning of End-Effectors for Micromanipulation

Precise rotational positioning of end-effectors under microscopy is crucial for robotic micromanipulation. However, the end-effector is presently limited to a fixed orientation, which is manually set before a given micromanipulation task, lacking accuracy and versatility of <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">in situ</i> reorientation. In this article, we present a unified framework for rotationally positioning the end-effector in three dimensions by establishing a general rotational model, developing a detection method within the limited field of view under microscopy, and designing a three-loop control strategy that adapts to different experimental requirements and model parameters. In experiments, a standard angled micropipette was used as the end-effector to verify the validity of the proposed methods. The performance was evaluated experimentally where the micropipette was robotically rotated to an arbitrarily desired orientation with an average orientation error less than <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$2^\circ$</tex-math></inline-formula> . In the experiments of sperm manipulation, the <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">in situ</i> micropipette orientation control capability improved the success rate of sperm immobilization and achieved dexterous robotic sperm orientation for facile aspiration.

P-215 Use of hyaluronan-based solution as an alternative to polyvinylpyrrolidone to improve blastulation in ICSI

Abstract Study question This study aimed to analyze whether hyaluronan as an alternative to polyvinylpyrrolidone (PVP) for sperm immobilization, aspiration, and injection into the ooplasm improves ICSI outcomes. Summary answer The use of hyaluronan solution as an alternative to PVP improves the blastocyst formation rate and good quality blastocyst formation rate compared to PVP. What is known already The PVP solution facilitates ICSI procedures, such as sperm immobilization, aspiration, and injection into the ooplasm. However, it has been reported that injection of a small amount of PVP along with the sperm into the ooplasm negatively affects subsequent embryo development. Hyaluronan is a natural component of the extracellular matrix of the cumulus-oocyte complex. Balaban et al. (2003) reported that hyaluronan can replace PVP during ICSI procedures in the early embryo transfer cycle without adversely affecting pregnancy outcomes. This study analyzed whether using hyaluronan-based solution for the ICSI procedure would improves blastocyst development compared with ICSI using PVP. Study design, size, duration This retrospective study analyzed clinical medical reports at Takahashi Women’s Clinic, Japan. We included 4002 oocytes retrieved from 411 patients under 39 years of age who underwent ICSI using autologous oocytes between December 2019 and August 2021. Of the oocytes used for ICSI, 1909 underwent sperm aspiration and injection into the ooplasm with hyaluronan (Sperm Slow; Origio), and 2093 oocytes with 7% PVP (NakaMedical). We did not perform preimplantation genetic testing-aneuploidy for any cycles. Participants/materials, setting, methods We used PVP droplets for sperm selection under 400× magnification in both groups. In hyaluronan-ICSI, the selected sperm were transferred to a hyaluronan droplet and washed three times. The sperm was then aspirated with hyaluronan, and ICSI was performed. In PVP-ICSI, all procedures were performed using PVP. The ICSI and embryo transfer outcomes were compared between hyaluronan-ICSI and PVP-ICSI by logistic regression analysis considering patient age, BMI, and basal level of anti-mullerian hormone. Main results and the role of chance Normal fertilization rates were 74.3% (1556/2093) in PVP-ICSI and 75.5% (1442/1909) in hyaluronan-ICSI. There was no significant difference in the normal fertilization rate between PVP-ICSI and hyaluronan-ICSI groups (p = 0.437, aOR:1.06, 95% CI: 0.92–1.22). We cultured 1323 2PN embryos in PVP-ICSI and 1237 2PN embryos in hyaluronan-ICSI until the blastocyst stage. Blastocyst formation rates were 48.1% for PVP-ICSI and 52.3% for hyaluronan-ICSI, and this difference was statistically significant (aOR, 1.20; 95% CI: 1.02–1.40; p = 0.024). Moreover, the good grade (Gardner criteria ≧BB) blastocyst formation rates were significantly higher in the hyaluronan-ICSI group (36.9% and 41.0%, aOR: 1.21, 95% CI: 1.03–1.42, p = 0.022). During the study period, we performed 163 and 169 cryo-thawed blastocyst transfer cycles in PVP-ICSI and hyaluronan-ICSI, respectively. The clinical pregnancy rate (50.9% vs. 54.4%, aOR: 1.19, 95% CI: 0.77–1.83, p = 0.443) and miscarriage rate (19.3% vs. 13.0%, aOR: 0.66, 95% CI: 0.3–1.44, p = 0.295) after embryo transfer were not significantly different between PVP-ICSI and hyaluronan-ICSI. Limitations, reasons for caution The study was conducted at a single IVF center, and the oocytes included in this study were collected from patients aged &amp;lt; 39 years. Embryo transfer result is based on ongoing pregnancy, while the live birth data for all pregnancies are not yet available. Wider implications of the findings Hyaluronan facilitates ICSI procedures such as sperm immobilization, aspiration, and injection. Moreover, the hyaluronan improves blastocyst development. The present study indicates that using hyaluronan as an alternative to PVP during the ICSI procedure is recommended. Trial registration number not applicable

O-044 ICSI in the lab: from vintage to AI

Abstract With the advent of in vitro fertilization (IVF) by Patrick Steptoe and Bob Edwards in the late 70s, the first conception outside of the human body resulted in the birth of Louise Brown. Although a terrific success, limitations of IVF surfaced, represented by the unexpected complete fertilization failure with suboptimal or dysfunctional spermatozoa. This prompted curiosity toward individual spermatozoa for a deeper understanding of its role aimed at enhancing the interaction between complementary gametes. Techniques were designed to manipulate the oocytes, such as stripping, partially digesting, or cracking the zona pellucida (ZP). These methods were palliative solutions to overcome fertilization failure and were often plagued by polyspermy. As a result, more direct approaches were implemented to overcome the ZP, such as subzonal injection (SUZI) that although more consistent, was still unable to overcome the shortcomings of dysfunctional spermatozoa. This laid the foundation for the utilization of ICSI that, whilst attempted by some investigators, became popular when Gianpiero Palermo serendipitously inserted one spermatozoon into the ooplasm during SUZI. Consistent fertilization then followed by injecting a cohort of oocytes by ICSI in SUZI cycles, and replacement of these embryos led to 4 pregnancies described in the first clinical ICSI report. To minimize oocyte damage, the procedure was further refined by inducing a deep invagination of the oolemma toward the 9 o’clock position, granting higher chances of post-injection survival. What set apart ICSI from other forms of ART was that any sperm sample, regardless of quality/quantity, would yield fertilization. Indeed, ICSI is the sole insemination method used with epididymal and testicular spermatozoa and has therefore revolutionized fertility treatment of azoospermic men. Indeed, even immotile testicular spermatozoa can still fertilize and yield successful pregnancies, albeit at a lower rate than their motile counterpart. Also, for these semen sources, aggressive sperm immobilization was introduced to enhance sperm membrane permeabilization and grant optimal fertilization results. Furthermore, ICSI has transformed the field of reproductive medicine by assisting other reproductive techniques, such as testing embryos for single gene defects to reduce the occurence of sperm DNA contamination, or overcoming the cryostress-induced changes of the ZP during cryopreservation allowing the oocyte to be fertilized at a higher rate. Oocyte cryopreservation now empowers women in their reproductive age to ordain their childbearing future. ICSI has proven to be the ultimate technique to overcome male infertility and has broadened its indication by yielding consistent fertilization and successful pregnancies in most circumstances, ensuring that men have the chance of fathering their own progeny. To date, ICSI is applied in several countries, and in some, is performed as the preferred/sole insemination method contributing to the birth of millions of babies worldwide. Thus far, no concerning differences have been seen in the health of ICSI versus standard IVF offspring, or even naturally conceived. In fact, it has been currently established that young adults of both genders born through ICSI retain their reproductive health. Despite its growing popularity, ICSI does not always succeed but still provides an invaluable platform to deepen our knowledge of gamete biology and helps to investigate/overcome some of the most severe and persistent forms of infertility. For example, combined with assisted gamete treatment, ICSI allows couples plagued by sperm-bound oocyte-activation-deficiency to achieve pregnancy. The need to increase access and curtail costs of reproductive care has led to the testing of automation in ART. This is also occurring with ICSI and to date, different automated modules have been proposed for oocyte denudation, sperm tracking, and robotic ICSI. Concurrently, there has been an interest in experimenting with artificial intelligence in the IVF laboratory to minimize human shortcomings and ensure that the best spermatozoon is chosen.

Використання полівінілалкоголю та полівінілпіролідону для підготовки деконсервованих еякульованих сперматозоїдів кнурів до запліднення методом ICSI

The aim of the study was to explore the effect of PVP (polyvinylpyrrolidone) and PVA (polyvinyl alcohol) media on deconserved ejaculated boar sperm and their preparation for artificial insemination to optimize biotechnological approaches. The studies used ejaculated cryopreserved sperm of a boar of the Myrhorod breed Dnipro 641. Genetic material was stored in the Bank of Genetic Resources of Animals IABG nnamed after M.V. Zubets NAAS for eight years. The sperm suspension was thawed in a water bath at +37 °C for 5 min until completely thawed. Separation of sperm from cryopreservative agent and diluent was performed using the swim up method in Sp-TALP medium. After the presence of sperm in the 10.0% solution of PVP for 10 min, motility decreased by 68.2% (P &lt; 0.05) and amounted to 3.4%, and after the next 10 min of incubation decreased to 1.4% (P &lt;0.01), which is 10 times lower than the initial mobility. In 10.0% of PVA mobility after 10 min of incubation decreased by 37.4% (P &lt;0.05) and amounted to 6.7%, and after 10 min decreased to 5.7% (P &lt; 0.01), which is 1.8 times lower than the initial mobility. It was found that in the case of 10.0% of PVP solution ejaculated deconserved boar sperm lose motility by 86.9% (P &lt;0.01) from the initial motility, which makes it impossible to select a suitable sperm for fertilization by ICSI (intracytoplasmic sperm injection). It is shown that 10.0% PVA solution can be used for immobilization of boar sperm, as it reduces motility by 46.7% (P &lt;0.01) of the initial sperm motility. It is proved that the mobility in the case of incubation of deconserved ejaculated boar sperm in 5.0% PVA solution decreases only by 28.0% (P &lt;0.05) from the initial, which is optimal when using cryopreserved boar sperm, material which are limited and convenient for the operator and safe for oocytes.

Evaluation of Antifertility Effects of Sperm Immobilization Factor from Escherichia coli on Male Mice

Earlier in our laboratory, sperm immobilization factor (SIF) isolated from Escherichia coli has shown admirable contraceptive efficacy in female mice and thus generated interest in evaluating the same as a potential candidate for male contraception. Keeping this in mind, the present study was aimed at exploitation of SIF as contraceptive agent in male mouse model after intra testis administration. For this, Balb/c mice were administered with different concentrations (10, 50, 100, 200, or 400µg) of SIF and sacrificed on days 3,7,14,21,30,45,60, and 90. After sacrifice, results in terms of change in body weight, tissue somatic indices, seminal parameters, histology, hematological parameters, lipid peroxidation, testosterone level, and cytokines level were evaluated. The weight profile and TSI of all experimental groups showed no significant change. In case of seminal parameters, 400μg of SIF led to azoospermia up to 90 days and tissue histology also revealed hypo-spermatogenesis. No significant change was observed in haematological parameters after SIF (400µg) administration however, a significant decrease was observed in testosterone levels. The oxidative status and cytokines profile was also affected after SIF (400µg) inoculation. Thus, when administered via intra testis route, SIF can lead to azoospermia and poses to be a potent and safe antifertility agent.

Antibacterial, Sperm Immobilization and Spermicidal Activities of Bacteriocins Produced by Vaginal Lactobacilli

Abstract Lactobacillus bacteriocin is an interesting compound playing a dual spermicidal/bacteriocidal role so that this compound can be developed as an agent that can be able to replace the unsafety chemical, nonoxynol-9. The present study aimed to investigate the antibacterial, sperm immobilization, and spermicidal activities of bacteriocins that were produced from vaginal lactobacilli. Nine of 932 cell-free supernatants exhibited potent antibacterial activities against the standard bacterial strains and the pathogenic Niesseria gonorrhoeae. Ion exchange-purified bacteriocins B84/7 F2 demonstrated the lowest minimum inhibitory concentration and the highest bacteriocin activity against the tested pathogens. The isolate B84/7 was then identified as Lactobacillus crispatus. The rapid killing activity was exerted within 15 minutes after being exposed to the tested bacteria, and also significantly inhibited the exponential growth of the tested pathogen. Among 48 semen of volunteers, the median time of sperm immobilization of ion-exchange purified B84/7 F2 bacteriocin (18.70 mg/mL) was 30.42 seconds. Within 60 seconds, spermicidal activities of the partial fractionated and ion-exchange purified B84/7 F2 at the undiluted concentration were 83.67 and 80.75%, respectively. Its activity will significantly decline at pH &gt; 8.0 or after being treated with alkaline and proteolytic enzymes. Two peptide bands of this bacteriocin were 7.08, and 8.81 kDa after being detected on a tricine SDS-PAGE gel. In-depth characteristics of this bacteriocin should be further investigated regarding the mode of action and purification. These findings can be applied to develop as the protecting agent of sexually transmitted infections especially gonorrhea, and unwanted pregnancy. Keywords: Antibacterial activity, Bacteriocins, Neisseria gonorrhoeae, Sperm immobilization, Spermicidal activity, Vaginal lactobacilli