Two laser-assisted hatching methods of embryos in ART: a systematic review and meta-analysis.

BACKGROUND: Laser-assisted hatching (LAH) stands as the predominant technique for removing the zona pellucida (ZP) in embryos, primarily consisting of two methods: drilling laser-assisted hatching (D-LAH) and thinning laser-assisted hatching (T-LAH). Presently, both methods have limitations, and their comparative efficacy for embryo implantation and clinical pregnancy remains uncertain. AIM: Evaluate the impact of D-LAH and T-LAH on clinical pregnancy rates within assisted reproductive technology (ART). METHODS: We systematically searched electronic databases including PubMed, Web of Science, and Cochrane Library until July 20, 2022. This study encompassed observational studies and randomized controlled trials (RCTs). A 95% confidence interval (CI) was utilized for assessing the risk ratio (RR) of pregnancy outcomes. The level of heterogeneity was measured using I2 statistics, considering a value exceeding 50% as indicative of substantial heterogeneity. RESULTS: The meta-analysis scrutinized 9 studies involving 2405 clinical pregnancies from D-LAH and 2239 from T-LAH. Findings suggested no considerable variation in the clinical pregnancy rates between the two techniques (RR = 0.93, 95% CI: 0.79-1.10, I2 = 71%, P = 0.41). Subgroup analyses also revealed no substantial differences. However, D-LAH exhibited a notably higher occurrence of singleton pregnancies compared to T-LAH (RR = 2.28, 95% CI: 1.08-4.82, I2 = 89%, P = 0.03). There were no noteworthy distinctions observed in other secondary outcomes encompassing implantation rate, multiple pregnancies, ongoing pregnancy, miscarriage, premature birth, and live birth. CONCLUSION: Both the primary findings and subgroup analyses showed no marked variance in clinical pregnancy rates between D-LAH and T-LAH. Therefore, patients with varying conditions should select their preferred LAH technique after assessing their individual situation. However, due to the restricted number of studies involved, accurately gauging the influence of these laser techniques on clinical outcomes is challenging, necessitating further RCTs and high-quality studies to enhance the success rate of ART. TRIAL REGISTRATION: PROSPERO: CRD42022347066.

Magnetic-Assisted Control of Eggs and Embryos via Zona Pellucida-Linked Nanoparticles.

Eggs and embryo manipulation is an important biotechnological challenge to enable positioning, entrapment, and selection of reproductive cells to advance into a new era of nature-like assisted reproductive technologies. Oviductin (OVGP1) is an abundant protein in the oviduct that binds reversibly to the zona pellucida, an extracellular matrix that surrounds eggs and embryos. Here, the study reports a new method coupling OVGP1 to magnetic nanoparticles (NP) forming a complex (NPOv). NPOv specifically surrounds eggs and embryos in a reversible manner. Eggs/embryos bound to NPOv can be moved or retained when subjected to a magnetic force, and interestingly only mature-competent eggs are attracted. This procedure is compatible with normal development following gametes function, in vitro fertilization, embryo development and resulting in the birth of healthy offspring. The results provide in vitro proof-of-concept that eggs and embryos can be precisely guided in the absence of physical contact by the use of magnets.

ZP2 cleavage blocks polyspermy by modulating the architecture of the egg coat.

Following the fertilization of an egg by a single sperm, the egg coat or zona pellucida (ZP) hardens and polyspermy is irreversibly blocked. These events are associated with the cleavage of the N-terminal region (NTR) of glycoprotein ZP2, a major subunit of ZP filaments. ZP2 processing is thought to inactivate sperm binding to the ZP, but its molecular consequences and connection with ZP hardening are unknown. Biochemical and structural studies show that cleavage of ZP2 triggers its oligomerization. Moreover, the structure of a native vertebrate egg coat filament, combined with AlphaFold predictions of human ZP polymers, reveals that two protofilaments consisting of type I (ZP3) and type II (ZP1/ZP2/ZP4) components interlock into a left-handed double helix from which the NTRs of type II subunits protrude. Together, these data suggest that oligomerization of cleaved ZP2 NTRs extensively cross-links ZP filaments, rigidifying the egg coat and making it physically impenetrable to sperm.

Mechanical Hatching as a Therapeutic Intervention for Improving Implantation Rate in a 32-Year-Old Female With Recurrent Implantation Failures: A Case Report.

This case report explores the application of mechanical hatching as a successful intervention in the treatment of primary infertility for a couple with a consanguineous marriage history and recurrent implantation failure. A 32-year-old female patient and her 37-year-old spouse, after six years of unsuccessful attempts to conceive, underwent multiple intrauterine insemination (IUI) and in vitro fertilization (IVF) and embryo transfer (ET) cycles without success. Normal parameters were observed in semen analysis and hormone tests for the male and female partners, respectively. Despite a series of failed assisted reproductive technology (ART) procedures, the implementation of mechanical hatching using partial zona dissection (PZD) pipettes led to a positive pregnancy outcome. The case underscores the potential efficacy of individualized approaches, specifically mechanical hatching, in addressing challenges associated with implantation failure, offering hope to couples facing infertility issues.

Tex46 knockout male mice are sterile secondary to sperm head malformations and failure to penetrate through the zona pellucida.

Each year, infertility affects 15% of couples worldwide, with 50% of cases attributed to men. It is assumed that sperm head shape is important for sperm-zona pellucida (ZP) penetration but research has yet to elucidate why. We generated testis expressed 46 (Tex46) knockout mice to investigate the essential roles of TEX46 in mammalian reproduction. We used RT-PCR to demonstrate that Tex46 was expressed exclusively in the male reproductive tract in mice and humans. We created Tex46-/- mice using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system and analyzed their fertility. Tex46 null spermatozoa underwent further evaluation using computer-assisted sperm analysis, light microscopy, and ultrastructural microscopy. We used immunoblot analysis to elucidate relationships between TEX46 and other acrosome biogenesis-related proteins. Mouse and human TEX46 are testis-enriched and encode a transmembrane protein which is conserved from amphibians to mammals. Loss of the mouse TEX46 protein causes male sterility primarily due to abnormal sperm head formation and secondary effects on sperm motility. Tex46 null spermatozoa morphologically lack the typical hooked sperm head appearance and fail to penetrate through the ZP. Electron microscopy of the testicular germ cells reveals malformation of the acrosomal cap, with misshapen sperm head tips and the appearance of a gap between the acrosome head and the nucleus. TEX46 is essential for sperm head formation, sperm penetration through the ZP, and male fertility in mice, and is a putative contraceptive target in men.

Zona pellucida removal resulted in a successful live birth: Report on a case with recurrent implantation failure due to embryonic bacteria contamination.

Key clinical message: In in vitro fertilization (IVF), laser offers several advantages. In this study, we employed laser to eliminate the zona pellucida of a contaminated embryo. This approach helps to rescue embryo with bacterial contamination, and improve embryo-endometrium interaction. Abstract: To present a case report on the removal of a contaminated zona pellucida from an embryo of patient with a history of recurrent implantation failure (RIF), which was followed by a successful live birth. We present the case of a 34-year-old patient with a history of 3 years of infertility who underwent in vitro fertilization. During the culture process, the embryos became contaminated, leading to three failed implantations. Despite the aneuploidy of the embryo and the implementation of a washing technique, the contamination persisted. In the final attempt, the contaminated zona pellucida was successfully removed using laser, followed by embryo transfer, resulting in a live birth. We provided detailed clinical information, including patient demographics, infertility history, ovarian response, evidence of bacterial contamination, embryo development, treatment protocols, and outcomes. Laser excision of the zona pellucida is a safe and effective method for addressing bacterial infection in embryos.

Sperm selection by zona adhesion improves assisted reproductive treatment outcomes.

BACKGROUND: Zona pellucida is one of the main selective barriers for the spermatozoa before reaching the oocyte. Using native zona in the sperm selection prior to Intracytoplasmic Sperm Injection (ICSI) has been proven effective but inconvenient approach in In Vitro Fertilisation (IVF) laboratory. The application of autologous solubilised zonae pellucidae in the sperm selection prior to ICSI has not been studied yet. OBJECTIVES: To compare the assisted reproductive treatment (ART) outcomes (implantation, pregnancy, live birth, and miscarriage rates) after ICSI performed with spermatozoa selected on their ability to adhere to immobilised solubilised zonae pellucidae and conventionally selected spermatozoa. MATERIALS AND METHODS: In total, 500 couples fulfilled the inclusion criteria and 368 of them were included in the study. After random allocation, 192 couples had spermatozoa selected by sperm-zona adhesion for ICSI (study group) and 176 patients underwent standard ICSI (control group). In the study group, patients' own zonae were acid solubilised and immobilised on petri dishes. The partner's motile spermatozoa were placed in the dishes and the adhered spermatozoa were used for ICSI. For the control group, the conventional sperm selection by morphological criteria was applied prior ICSI. All women underwent frozen ET with euploid embryos. Chi square test was used to compare the data. RESULTS: The sperm selection by zona adhesion resulted in significantly higher implantation rate (50.4% vs. 37.0%, p = 0.003), clinical pregnancy rate (43.8% vs. 33.3%, p = 0.018) and live birth rate (38.0% vs. 25.9%, p = 0.004) and significantly lower incidence of miscarriage (11.3% vs. 22.2%, p = 0.044) in comparison to the conventional method of the sperm selection. DISCUSSION AND CONCLUSION: The application of solubilised zonae pellucidae in the sperm selection for ICSI benefits ART outcomes in couples with unexplained infertility. Moreover, sperm-zona selection significantly reduces the risk of miscarriages.

Immunocontraceptive Efficacy of Native Porcine Zona Pellucida (pZP) Treatment of Nevada's Virginia Range Free-Roaming Horse Population.

In North America, range constraints due to burgeoning development increasingly encroach on wild horse habitat and necessitate effective but humane reproductive management. The largest free-roaming wild horse fertility control program by population (>3500) and territory size (≈300,000 acres) is located within Nevada's Virginia Range. Data from a field study investigated porcine zona pellucida (pZP) immunocontraception via remote dart delivery to mares in this population. Analyses aimed to measure efficacy by treatment effects on annual birth rates and population demographics and to evaluate treatment frequency and season against these variables. Analyses included mares' monthly data (January 2019-December 2022; 48 months), characterized by cumulative vaccination numbers subset into four classifications considering the vaccine as having no loss of efficacy or a loss within a 6-, 12-, and 18-month period post vaccination; from foaling data, the likelihood of being in foal and of conceiving in that month; and from age, as mature or immature (<1 years-old). A downward foaling rate and trend in the numbers of mature mares, descriptively presented at monthly intervals, showed markedly declining annual seasonal breeding peaks, with no observed change in foaling season or duration. Within four years, population coverage surpassed 70% and was associated with a 58% reduction in foaling, with only a 10% conception rate. Vaccinated mares increased proportionally: assuming a 12-month decay rate, the system reached stability at an average ≈1.0 vaccination/mare/year, providing a robust recommendation for treatment frequency contributing to best management practices.

Sperm Selection Using Zona Pellucida-Binding Enhanced Embryo Morphokinetic and Clinical Outcomes in ICSI: A Sibling Oocytes Study.

The objective was to investigate the embryo morphokinitics using a time-lapse monitoring (TLM) system and assessment of clinical outcomes following intracytoplasmic sperm injection (ICSI) with zona pellucida (ZP)-bound sperm selection and conventional methods. A total of 371 metaphase II (MII) oocytes from 50 ICSI cycles were studied. Sibling oocytes were randomly divided into control (n = 199) and ZP-bound group (n = 172). All resulting zygotes were cultured and monitored in the TLM system up to Day 3 after ICSI. Fertilization rate, early embryo development, and clinical outcomes were evaluated. No significant differences were found in fertilization rate, time-lapse qualitative and quantitative measures, pronuclear fading time (PNF) t2, t3, t4, t5, t6, and t7 (times of cleavage to 2, 3, 4, 5, 6, and 7 cells), respectively. However, the t8 (time of cleavage to eight cells) and cc3 (duration of third cell cycle) revealed a significant difference between control and ZP-bound groups (p < .05). A significant difference between the two groups (p < .05) in the rates of Grade A embryos (according to Basile algorithm), chemical pregnancy, clinical pregnancy, and implantation was observed. Sperm selection using biological materials, such as ZP, improved both embryo quality and pregnancy outcomes, despite not affecting the early embryo development and morphokinetic parameters up to t8. This prospective randomized sibling oocyte trial was registered in October 2020 to January 2022 (IRCT20200705048021N1).

Embryological characteristics and clinical outcomes of oocytes with different degrees of abnormal zona pellucida during assisted reproductive treatment.

Abnormalities in the zona pellucida (ZP) adversely affect oocyte maturation, embryo development and pregnancy outcomes. However, the assessment of severity is challenging. To evaluate the effects of different degrees of ZP abnormalities on embryo development and clinical outcomes, in total, 590 retrieval cycles were scored and divided into four categories (control, mild, moderate and severe) based on three parameters: perivitelline space, percentage of immature oocytes and percentage of oocytes with abnormal morphology. As the severity of abnormal ZP increased, both the number of retrieved oocytes and mature oocytes decreased. The fertilization rate did not differ significantly among groups. The rates of embryo cleavage and day-3 high-quality embryos in the mild group and the moderate group did not vary significantly between the two groups but were significantly higher than those in the severe group. The blastulation rates of the abnormal ZP groups were similar; however, they were lower than those of the control group. Moreover, the cycle cancellation rate of the severe abnormal ZP group was as high as 66.20%, which was significantly higher than that of the other three groups. Although the rates of cumulative clinical pregnancy and live births were lower than those in the control group, they were comparable among the abnormal ZP groups. There were no differences in the neonatal outcomes of the different groups. Together, ZP abnormalities show various degrees of severity, and in all patients regardless of the degree of ZP abnormalities who achieve available embryos, there will be an opportunity to eventually give birth.

N-Glycosylation Site in the Middle Region Is Involved in the Sperm-Binding Activity of Bovine Zona Pellucida Glycoproteins ZP3 and ZP4.

Mammalian fertilization is a species-selective event that involves a series of interactions between sperm proteins and the oocyte's zona pellucida (ZP) glycoproteins. Bovine ZP consists of three glycoproteins: bZP2, bZP3, and bZP4. In our previous study, we demonstrated that bovine sperm binds to plastic wells coated with recombinant bZP4 and identified that the N-terminal domain and the middle region of bZP4 are critical for sperm-binding activity. Here, we investigated the sperm-binding site in the middle region (residues 290 to 340) of bZP4, which includes the hinge region. We showed that bovine sperm binds to bZP4's middle region in a species-selective manner. We mapped the function of bZP4's middle region to its N-glycosylation site at Asn-314 using several recombinant mutated proteins. Moreover, we showed that mutations of the N-glycosylation sites at Asn-314 close to the hinge region and Asn-146 of the hinge region of bZP4 and bZP3, respectively, reduced the sperm-binding activity of the complex of the bZP3 (from 32 to 178) and bZP4 (from 136 to 464) fragments. Together, these results suggest that ZP's middle regions of bZP3 and bZP4 form one of the sperm-binding sites of bovine ZP.

Intracellular fraction of zona pellucida protein 3 is required for the oocyte-to-embryo transition in mice.

In oocyte biology, the zona pellucida has long been known to operate three extracellular functions downstream of the secretory pathway, namely, encasing the oocytes in ovarian follicles, mediating sperm-oocyte interaction, and preventing premature embryo contact with oviductal epithelium. The present study uncovers a fourth function that is fundamentally distinct from the other three, being critical for embryonic cell survival in mice. Intriguingly, the three proteins of the mouse zona pellucida (ZP1, ZP2, ZP3) were found abundantly present also inside the embryo 4 days after fertilization, as shown by mass spectrometry, immunoblotting, and immunofluorescence. Contrary to current understanding of the roles of ZP proteins, ZP3 was associated more with the cytoskeleton than with secretory vesicles in the subcortical region of metaphase II oocytes and zygotes, and was excluded from regions of cell-cell contact in cleavage-stage embryos. Trim-away-mediated knockdown of ZP3 in fertilized oocytes hampered the first zygotic cleavage, while ZP3 overexpression supported blastocyst formation. Transcriptome analysis of ZP3-knockdown embryos pointed at defects of cytoplasmic translation in the context of embryonic genome activation. This conclusion was supported by reduced protein synthesis in the ZP3-knockdown and by the lack of cleavage arrest when Trim-away was postponed from the one-cell to the late two-cell stage. These data place constraints on the notion that zona proteins only operate in the extracellular space, revealing also a role during the oocyte-to-embryo transition. Ultimately, these data recruit ZP3 into the family of maternal factors that contribute to developmental competence of mouse oocytes.

The cell volume-regulatory glycine transporter GLYT1 is activated following metallopeptidase-mediated detachment of the oocyte from the zona pellucida.

Independent cell volume regulation is first acquired by the oocyte in two steps that occur during meiotic maturation: (1) activation of the glycine transporter GLYT1 (Slc6a9) that mediates the intracellular accumulation of glycine to provide osmotic support in the mature egg and early preimplantation embryo, and (2) release of the oocyte from the strong attachment to its rigid extracellular matrix shell, the zona pellucida (ZP). It was recently shown that oocyte-ZP detachment requires metallopeptidase activity that is proposed to cleave transmembrane ZP proteins connecting the oocyte to the ZP. It is unknown, however, how GLYT1 is activated. We hypothesized that oocyte-ZP detachment precedes and may be required for GLYT1 activation. In identically treated pools of oocytes, oocyte-ZP detachment occurred ~20 min before GLYT1 activation. In individual oocytes, GLYT1 activity was detected only in those that were mostly or fully detached. Blocking detachment using previously validated small molecule metallopeptidase inhibitors partly suppressed GLYT1 activation. However, removal of the ZP did not accelerate GLYT1 activation. This indicates that oocyte-ZP detachment or cleavage of transmembrane ZP proteins may be required for GLYT1 to become fully activated, or alternatively that metallopeptidase activity independently affects both detachment and GLYT1 activation.

Novel image analyser-assisted morphometric methodology offer unique opportunity for selection of embryos with potential for implantation.

BACKGROUND: Previous studies looked into the connections between pregnancy and the Zona Pellucida (ZP) thickness and Zona Pellucida Thickness Variation (ZPTV), as well as the embryo's radius, circumference, perimeter and global symmetry. However, no research has linked embryo implantation and pregnancy to the percentage of ZP thinning, the reduction in ooplasm volume, and the increase in perivitelline space (PVS) volume. Our objective is to correlate the percentage of ZP thinning, the percentage of ooplasm volume shrinkage and the percentage of PVS increase to the implantation. These data will be used for embryo selection as well as it can be put into a software that will assist embryo selection. MATERIALS AND METHODS: Retrospective study included 281 patients, all of them had 2 embryos transferred, 149 patients got pregnant with two gestation sacs and 132 patients did not get pregnant. All of the transferred embryos had the ZP thickness measured several times from time of ICSI till Embryo Transfer (ET), the ooplasm volume was calculated from time of ICSI till two Pronuclei (2PN) fading and the PVS was calculated from the ICSI time till the 2PN fading. RESULTS: The first characteristic is the change in the average ZP thickness that decreased by 32.7% + 5.3% at 70 h for the implanted embryos (Group 1) versus 23.6% + 4.8% for non-implanted embryos (Group 2) p = 0.000. The second characteristic is the average reduction in the volume of the ooplasm which is 20.5% + 4.3% in Group 1 versus 15.1% + 5.2% in Group 2, p = 0.000. The third characteristic is the increase in the volume of the PVS which was 38.1% + 7.6% in Group 1 versus 31.6% + 9.7% in Group 2 p = 0.000. CONCLUSION: The implanted embryos showed higher percent of ZP thinning, higher percent of ooplasm reduction and higher percent of PVS increase.

Comparative studies on ovary and serum hormone levels in silver and blue foxes and their hybrids.

The silver and blue fox appear closely related despite their belonging to different genera, and the male and female fox hybrids are completely sterile in mating inter se or back to either of the parental groups. In the present study, we compared ovarian morphology and serum hormone concentrations among silver foxes, blue foxes, HSBs (silver fox male × blue fox female hybrids) and HBSs (blue fox male × silver fox female hybrids) during the pre-breeding and the breeding period. We found that either the fertile blue and silver foxes or the infertile hybrid foxes showed typical characteristics of ovary, with primordial, primary, secondary, antral and Graafian follicles. The diameters and quantities of different follicle classes in fertile foxes were greater than that in hybrids during the two period, while the thickness of zona pellucida (ZP) represented an opposite trend (P < 0.05). The relative weight of bilateral ovaries in fertile foxes was greater than that in hybrids during the pre-breeding period, while mean serum FSH represented an opposite trend during the breeding period (P < 0.05). The mean serum estradiol in fertile foxes was greater than that in HBS foxes during the pre-breeding period, and greater than hybrids during the breeding period (P < 0.05). These findings indicated that in the infertile hybrid foxes, lower estradiol levels produced by lesser and smaller antral follicles, thicker ZP during the two period, higher FSH levels during the breeding period, could be the basis of interpretations in cases of female fox hybrid infertility.

Ovastacin: An oolemma protein that cleaves the zona pellucida to prevent polyspermy.

Monospermy occurs in the process of normal fertilization where a single sperm fuses with the egg, resulting in the formation of a diploid zygote. During the process of fertilization, the sperm must penetrate the zona pellucida (ZP), the outer layer of the egg, to reach the egg's plasma membrane. Once a sperm binds to the ZP, it undergoes an acrosomal reaction, which involves the release of enzymes from the sperm's acrosome that help it to penetrate the ZP. Ovastacin is one of the enzymes that is involved in breaking down the ZP. Studies have shown that ovastacin is necessary for the breakdown of the ZP and for successful fertilization to occur. However, the activity of ovastacin is tightly regulated to ensure that only one sperm can fertilize the egg. One way in which ovastacin helps to prevent polyspermy (the fertilization of an egg by more than one sperm) is by rapidly degrading the ZP after a sperm has penetrated it. This makes it difficult for additional sperm to penetrate the ZP and fertilize the egg. Ovastacin is also thought to play a role in the block to polyspermy, a mechanism that prevents additional sperm from fusing with the egg's plasma membrane after fertilization has occurred. In summary, the role of ovastacin in monospermic fertilization is to help ensure that only one sperm can fertilize the egg, while preventing polyspermy and ensuring successful fertilization.

Immunogenicity and contraceptive efficacy of plant-produced putative mouse-specific contraceptive peptides.

Rodent population control through contraception requires species-specific oral contraceptive vaccines. Therefore, in this study, we produced putative mouse-specific contraceptive peptides, mZP2 (from oocyte) and mIzumo1 (from sperm), in plants using Agrobacterium-mediated transient expression. Peptides were produced separately in Nicotiana benthamiana using constructs encoding antigens containing three copies of each peptide. We also determined the immunogenicity and contraceptive effects of the plant-produced antigens in female BALB/c mice. Mice immunized subcutaneously with a relatively low amount of antigen (5 µg/dose of each peptide in a mixture) showed systemic immune responses against mZP2-3 and mIzumo1-3 antigens. Moreover, the mean litter size of mice treated with the plant-produced antigens was reduced by 39% compared to that of the control mice. Notably, there was a significant negative correlation between the number of pups born and individual antibody levels against both antigens. Immunofluorescence assays demonstrated the binding of induced antibodies to the oocytes of BALB/c and wild-type mice in vivo and in vitro, respectively. Our study demonstrate the feasibility of producing small contraceptive peptides in plants that can be further used to develop oral contraceptive vaccines against mouse populations.

Viscoelastic Properties of Zona Pellucida of Oocytes Characterized by Transient Electrical Impedance Spectroscopy.

The success rate in vitro fertilization is significantly linked to the quality of the oocytes. The oocyte's membrane is encapsulated by a shell of gelatinous extracellular matrix, called zona pellucida, which undergoes dynamic changes throughout the reproduction cycle. During the window of highest fertility, the zona pellucida exhibits a softening phase, while it remains rigid during oocyte maturation and again after fertilization. These variations in mechanical properties facilitate or inhibit sperm penetration. Since successful fertilization considerably depends on the state of the zona pellucida, monitoring of the hardening process of the zona pellucida is vital. In this study, we scrutinized two distinct genetic mouse models, namely, fetuin-B wild-type and fetuin-B/ovastacin double deficient with normal and super-soft zona pellucida, respectively. We evaluated the hardening with the help of a microfluidic aspiration-assisted electrical impedance spectroscopy system. An oocyte was trapped by a microhole connected to a microfluidic channel by applying suction pressure. Transient electrical impedance spectra were taken by microelectrodes surrounding the microhole. The time-depending recovery of zona pellucida deflections to equilibrium was used to calculate the Young's modulus and, for the first time, absolute viscosity values. The values were obtained by fitting the curves with an equivalent mechanical circuit consisting of a network of dashpots and springs. The observer-independent electrical readout in combination with a fitting algorithm for the calculation of the viscoelastic properties demonstrates a step toward a more user-friendly and easy-to-use tool for the characterizing and better understanding of the rheological properties of oocytes.

Does Better Post-Thaw Motility of Dog Sperm Frozen with CLC Mean Better Zona Pellucida Binding Ability?

Even though the search for methods improving cryopreservation of canine spermatozoa led to an improvement of post-thaw quality, fertilizing results after insemination with frozen-thawed semen are still not satisfying. In this study, we focused on modification of spermatozoa membrane fluidity and investigated whether kinematic parameters as assessed by computer-assisted semen analyzer (CASA) can be improved. The primary aim of our study was to investigate whether the use of cholesterol-loaded cyclodextrins (CLC; 0.5 mg, 1 mg, 2 mg) and 2-Hydroxypropyl-ß-cyclodextrin (HBCD; 1 mg) positively influence capacitation status as examined by tyrosinphosphorylation, cholesterol efflux and zona binding assay (ZBA) of spermatozoa. The use of 0.5 mg of CLC increased the percentage of motile, progressive and rapid spermatozoa compared to the control. Addition of HBCD decreased motility and progressive motility of spermatozoa and the population with rapid movement in comparison to the control. The percentage of live spermatozoa without efflux of cholesterol compared to the control was increased when extender with 0.5 mg of CLC was used. There was no change in capacitation status. The zona binding ability of spermatozoa was significantly lower in the group with 0.5 mg of CLC than in the control. In conclusion, these results suggest that improvement of kinematic parameters does not necessarily coincide with better zona pellucida binding ability of spermatozoa.

The role of spermatozoa-zona pellucida interaction in selecting fertilization-competent spermatozoa in humans.

Human fertilization begins when a capacitated spermatozoon binds to the zona pellucida (ZP) surrounding a mature oocyte. Defective spermatozoa-ZP interaction contributes to male infertility and is a leading cause of reduced fertilization rates in assisted reproduction treatments (ARTs). Human ejaculate contains millions of spermatozoa with varying degrees of fertilization potential and genetic quality, of which only thousands of motile spermatozoa can bind to the ZP at the fertilization site. This observation suggests that human ZP selectively interacts with competitively superior spermatozoa characterized by high fertilizing capability and genetic integrity. However, direct evidence for ZP-mediated sperm selection process is lacking. This study aims to demonstrate that spermatozoa-ZP interaction represents a crucial step in selecting fertilization-competent spermatozoa in humans. ZP-bound and unbound spermatozoa were respectively collected by a spermatozoa-ZP coincubation assay. The time-course data demonstrated that ZP interacted with a small proportion of motile spermatozoa. Heat shock 70 kDa protein 2 (HSPA2) and sperm acrosome associated 3 (SPACA 3) are two protein markers associated with the sperm ZP-binding ability. Immunofluorescent staining indicated that the ZP-bound spermatozoa had significantly higher expression levels of HSPA2 and SPACA3 than the unbound spermatozoa. ZP-bound spermatozoa had a significantly higher level of normal morphology, DNA integrity, chromatin integrity, protamination and global methylation when compared to the unbound spermatozoa. The results validated the possibility of applying spermatozoa-ZP interaction to select fertilization-competent spermatozoa in ART. This highly selective interaction might also provide diagnostic information regarding the fertilization potential and genetic qualities of spermatozoa independent of those derived from the standard semen analysis.