Polyspermy Rates Research Articles

Nuclear maturation kinetics and in vitro fertilization of immature bovine oocytes injected into pre-ovulatory follicles

The maturation kinetics and in vitro fertilization of immature bovine oocytes injected by the intra-follicular oocyte injection (IFOT) technique into pre-ovulatory follicles of previously synchronized cows were evaluated. In Experiment 1, grade I, II and III cumulus-oocyte complexes (COCs) were randomly distributed to one of three Groups: Matvitro22 (COCs matured in vitro for 22 h), MatFol20 and MatFol28 (COCs matured in vivo after being injected into a pre-ovulatory follicle of previously synchronized cows for 19.8 ± 0.1 h and 28.3 ± 0.1 h, respectively). Cows received 12.5 mg of LH (Lutropin, Bioniche, Canada) at the time of IFOT in the MatFol20 Group or 10 h after IFOT in the MatFol28 Group. MatFol20 and MatFol28 COCs were aspirated approximately 20 h after the LH injection for nuclear maturation kinetics and recovery rate assessment. In Experiment 2, grade I, II, and III COCs were randomly distributed into two Groups: Matvitro22 Group, COCs were matured and fertilized in vitro, and MatFol20 Group, COCs were matured as in the MatFol20 Group in Experiment 1, but COCs were fertilized in vitro. Putative zygotes were classified as fertilized, unfertilized or polyspermic. In Experiment 1, the recovery rate was lower (P < 0.001) in the MatFol20 Group (52.9%, 91/172) compared with MatFol28 (72.9%, 113/155). Rate of oocytes in germinal vesicle stage, metaphase I, anaphase I and telophase I were similar among Groups. However, oocytes matured in vivo for 28.3 h had lower rate of metaphase II (P = 0.001) and greater rates of degenerated (P = 0.001) and parthenogenetically activated (P = 0.001) oocytes. In experiment 2, the rates of polyspermy and degenerated were similar between Groups. However, the rate of fertilized oocytes was greater (P = 0.05) in oocytes in the MatFol20 Group. It is concluded that oocyte in vivo maturation for 19.8 h after IFOT does not compromise the nuclear maturation kinetics and increases in vitro fertilization rates. However, the extra 10 h of intra-follicular incubation time decreased oocyte viability.

Early Cumulus Cell Removal Increases Cumulative Live Birth Rate While Having No Negative Effect on the Malformation Rate in in Vitro Fertilization: A Propensity Score-Matched Cohort Study

Background: Early cumulus cell removal (ECCR) in IVF may benefit from reducing potential detrimental exposure to free oxygen radicals (ROS) and other metabolic products which come from spermatozoa. While the other studies showed that EECR may be detrimental to embryo development and may break the important cross-talk between oocyte and cumulus cells during early embryos development. The effect of ECCR remains controversial to date. The aim of this study was to investigate the efficacy and safety of ECCR during human in vitro fertilization (IVF). Methods: A retrospective analysis was performed between January 2011 and December 2019. The study enrolled 1,131 couples who underwent IVF treatment with ECCR. After propensity score matching at a 1:1 ratio, 1,131 couples who underwent overnight coincubation of gametes were selected. The main outcome measure was the cumulative live birth rate. Secondary outcome measures included the cumulative pregnancy rate, polyspermy rate, available embryo rate, miscarriage rate, malformation rate, time to live birth, and oocyte-to-baby rate. Findings: There were no significant differences found between the two groups in the polyspermy rate, available embryo rate, miscarriage rate, time to live birth, oocyte-to-baby rate, and neonatal congenital anomalies rate. The results of the study showed that ECCR was associated with a significantly higher cumulative live birth rate and cumulative pregnancy rate, along with a significantly lower fertilization rate. Interpretation: ECCR tended to confer increased cumulative live birth rate and had no negative effect on the neonatal malformation rate. Funding Information: This work was supported by the Shanghai Public Health Bureau. Declaration of Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics Approval Statement: The protocol of this study was reviewed and approved by the Institutional Reviews Boards and Ethics Committee of Shanghai First Maternity and Infant Hospital, China on August 15, 2019 (reference number: 2019-57).

Excess polyspermy reduces the ability of porcine oocytes to promote male pronuclear formation after in vitro fertilization.

Male pronucleus (MPN) formation is a very important physiological event during fertilization, which affects in vitro production of transferrable embryos. The aim of this study was to find out the correlation between the number of penetrated sperm and the occurrence of failure of MPN formation in porcine oocytes. In vitro matured porcine oocytes were fertilized in vitro with frozen epididymal sperm. Two different frozen sperm lots were tested in this study, which were different in terms of polyspermy rates. The numbers and the status of penetrated sperm in oocytes were evaluated 10 h after insemination. Under high polyspermy condition, the polyspermy rate was 83.5% with an average mean of 3.5 sperms per penetrated oocyte, whereas the percentage of polyspermy was 65.5% with an average mean of 2.4 sperms per penetrated oocyte under moderate polyspermic condition. Correlation analysis revealed a negative correlation between the number of penetrated sperm and their MPN formation percentage both in the sperm lot of high polyspermy (R = −0.560, p < 0.05) and in the sperm lot of moderate polyspermy (R = −0.405, p < 0.05) which suggests that penetration of excessive spermatozoa disables the oocyte cytoplasm to promote MPN formation.

Addition of exogenous proteins detected in oviductal secretions to in vitro culture medium does not improve the efficiency of in vitro fertilization in pigs

This work was designed to study whether HSP70-1A, HSP90α, ezrin or PDI4, proteins previously identified in porcine oviductal secretions, have a role in zona pellucida (ZP) resistance to enzymatic digestion, in vitro fertilization (IVF) and sperm viability. In vitro matured porcine cumulus oocyte complexes were denuded and i) incubated for 1 h in TALP medium supplemented or not with each exogenous oviductal protein and in presence or absence of heparin to assess ZP digestion time by pronase; and ii) inseminated with fresh ejaculated boar spermatozoa in medium supplemented or not with each exogenous oviductal protein to assess their effect on fertilization results. Finally, spermatozoa were incubated in Tyrode’s medium (0, 1 and 20 h) supplemented or not with HSP-701A, HSP-90α or ezrin, to assess simultaneously sperm viability and acrosome status by means of flow cytometry. Although all proteins increased the ZP digestion time, this increase was lower than 1 min, being ezrin the protein with a stronger effect. Presence of heparin in the medium reinforced the ZP hardening effect of ezrin and HSP-701A up to one more min, but not HSP-90α nor PDI4. Sperm penetration, but not IVF efficiency, increased when gametes were cocultured in medium containing PDIA4 whereas sperm penetration and polyspermy rates decreased in presence of ezrin and HSP proteins. This reduction was not the result of a detrimental effect of proteins on sperm viability or acrosome reaction. In conclusion, addition of exogenous proteins detected in oviductal secretions to artificial media does not reproduce the effect of adding such secretions nor improve the final efficiency of the porcine IVF system.

Perinatal and neonatal outcomes of pregnancies after early rescue intracytoplasmic sperm injection in women with primary infertility compared with conventional intracytoplasmic sperm injection: a retrospective 6-year study

BackgroundEarly rescue intracytoplasmic sperm injection (ICSI) has been used in clinic as appropriate currently. While the outcomes of children born after this method were not well assessed. The purpose of this study was to evaluate the effect of early rescue ICSI on women with primary infertility.MethodsFresh embryo transfer cycles after rescue (n = 214) and conventional (n = 546) ICSI were retrospectively evaluated from women with primary infertility who underwent their first assisted reproductive technology cycles at our center in 2012–2017. The conventional ICSI group was subdivided into ICSI-1 (semen suitable for in vitro fertilization, IVF) and ICSI-2 (poor semen quality) to minimize bias from differences in semen quality. Pregnancy, delivery and neonatal outcomes were compared between groups.ResultsThere was a higher rate of polyspermy and a lower rate of top-quality embryos (TQE) on day 3 for oocytes subject to rescue ICSI compared with conventional ICSI. This reduced the total number of TQE and the number of TQE transferred in the rescue ICSI group. There was no significant difference between groups in clinical pregnancy, ongoing pregnancy, early miscarriage and live birth. For pregnant women, gestational age, route of delivery, risk of preterm birth and gestational diabetes mellitus were also comparable. Neonatal outcomes including sex ratio, birth weight, neonatal intensive care unit admission and birth defects were also similar after rescue and conventional ICSI. Moreover, no differences were observed with the different ICSI subgroups.ConclusionsFor women with primary infertility who have a high risk of IVF fertilization failure (FF), rescue ICSI provides a safe and efficient alternative to minimize FF after initial IVF, but results in fewer TQE on day 3.

Correlation between insemination dose and bovine sperm traits on the in vitro embryo production outcomes

In the present study we aimed to test the best insemination dose for in vitro embryo production (IVEP) and to correlate sperm traits in bovine. In vitro matured oocytes were inseminated with three different sperm concentrations of the same bull: G1 (1*106 ), G2 (2*106 ) and G3 (4*106 ) sperm/mL. At 18 h post-insemination (hpi), presumptive zygotes [G1 (n=114), G2 (n=139) and G3 (n=136)] were stained to evaluate the pronuclei numbers, or continued to in vitro culture [G1 (n=102), G2 (n=111) and G3 (n=106)]. Sperm kinetics were analyzed using Computer-Assisted Semen Analysis (CASA). Sperm plasma membrane, acrosome integrity and mitochondrial activity were analyzed using fluorescent probes. In vitro fertilization (IVF) and IVEP data were compared using chisquare (P 0.05) among the groups. In G3, the polyspermy rate was the highest (7.4%; P 0.05) between G1 (0%) and G2 (0%). In G1, the early blastocyst rate was the highest (7.8%; P 0.05) between G2 (1.8%) and G3 (0.9%). The IVF efficiency and total blastocyst rates were positively correlated with curvilinear velocity (VCL) (r≃+1; P<0.05). We concluded that the reduction of insemination dose may negatively affect embryo development and VCL may be used as a parameter to improve the IVEP outcomes.

Coculture of porcine cumulus-oocyte complexes with porcine luteal cells during IVM: effect on oocyte maturation and embryo development.

Coculture with somatic cells is an alternative to improve suboptimal invitro culture conditions. In pigs, IVF is related to poor male pronuclear formation and high rates of polyspermy. The aim of this study was to assess the effect of a coculture system with porcine luteal cells (PLCs) on the IVM of porcine cumulus-oocyte complexes (COCs). Abattoir-derived ovaries were used to obtain PLCs and COCs. COCs were matured invitro in TCM-199 with or without the addition of human menopausal gonadotrophin (hMG; C+hMG and C-hMG respectively), in coculture with PLCs from passage 1 (PLC-1) and in PLC-1 conditioned medium (CM). In the coculture system, nuclear maturation rates were significantly higher than in the C-hMG and CM groups, but similar to rates in the C+hMG group. In cumulus cells, PLC-1 coculture decreased viability, early apoptosis and necrosis, and increased late apoptosis compared with C+hMG. PLC-1 coculture also decreased reactive oxygen species levels in cumulus cells. After IVF, monospermic penetration and IVF efficiency increased in the PLC-1 group compared with the C+hMG group. After invitro culture, higher blastocysts rates were observed in the PLC-1 group. This is the first report of a coculture system of COCs with PLCs. Our model could be an alternative for the conventional maturation medium plus gonadotrophins because of its lower rates of polyspermic penetration and higher blastocysts rates, key issues in porcine invitro embryo production.

186 Protective effect of resveratrol on invitro fertilization of ovine oocytes matured under cadmium exposure

Cadmium (Cd) is one of the most toxic environmental and industrial heavy metals, with adverse effect on female reproduction. Earlier invitro studies have demonstrated that exposure to Cd during IVM impairs oocyte fertilization through the induction of oxidative stress (Martino et al. 2017 Reprod. Toxicol. 69, 132-145). The aim of this study was to evaluate the effect of supplementing IVM medium with resveratrol (Res), a phytoalexin with antioxidant activity, on IVF of ovine oocytes matured under Cd exposure and to evaluate whether this effect could be mediated by alterations of cortical granules (CGs) distribution and/or intracellular levels of reactive oxygen species (ROS). Cumulus-oocyte complexes collected from slaughterhouse-derived ovaries of prepubertal ewes were invitro matured with 0μM CdCl2 (control), 2μM CdCl2 (Cd group), or 2μM CdCl2 + 1μM Res (Cd-Res). Groups of MII oocytes were invitro fertilized with ram semen (Bogliolo et al. 2011 Reprod. Fertil. Dev. 23, 809-817). After 16h, oocytes were fixed with absolute ethanol, stained with Hoechst 33342, and classified as follows: normally fertilized (two pronuclei), polyspermic (more than two pronuclei), and abnormally fertilized (asynchronous pronucleus formation). Levels of ROS and distribution of CG in MII oocytes (n=25 each group) were evaluated by confocal laser scanning microscopy after staining with 2’,7’dichlorodihydrofluorescein diacetate and Alexa Fluor 488-conjugated lectin peanut agglutinin, respectively (Jiao et al. 2013 PLoS One 8, 58018). Data of fertilization and CGs distribution were analysed by Chi-squared test and ROS levels (mean±standard error) by Kruskal-Wallis test with STATA\IC 11.0 (Stata Corp.). Oocytes matured in presence of Res had higher normal fertilization and lower polyspermy rates than those matured without Res (Table 1). Levels of ROS in oocytes were lower in the Cd-Res group than in the Cd group (74.4±1.3 vs. 122.4±1.5 pixel/oocyte; P&amp;lt;0.05), and similar to those of the control (71.1±2.3 pixel/oocyte). The ratio of oocytes of Cd-Res group with CGs normally distributed in the peripheral region was higher than that of Cd group (91.3% vs. 65.2%; P&amp;lt;0.05) and comparable to that of control oocytes (95.8%). Collectively, our results showed that resveratrol protects oocytes from Cd-induced alteration of normal fertilization by preventing oxidative stress and improving the major mechanism responsible for blockage of polyspermic fertilization. Table 1.Effect of resveratrol on IVF rate of oocytes matured under cadmium exposure Group No. of total oocytes No. of MII oocytes No. of fertilized oocytes (%) Normal Polyspermy Abnormal Control 136 105 (77.2) 59 (56.2)a 23 (21.9)a 6 (5.7) Cd 146 106 (72.6) 39 (36.8)b 38 (35.8)b 7 (6.6) Cd-Res 114 88 (77.2) 47 (53.4)a 16 (18.2)a 7 (7.9) a,bValues in the same column with different superscripts differ significantly (P&amp;lt;0.05). This project was supported by FDS 2016 (CUP J86C18000780005).

119 Short spermatozoa-oocyte co-incubation improves outcomes of IVF in sheep

The assisted reproductive technique IVF is routinely applied in humans and large animals, both to boost reproductive performance and also for basic research. Despite its value, IVF has seen very little progress in the last two decades and relies on established paradigms, such as overnight sperm-egg co-incubation. However, the long exposure of oocytes to spermatozoa in a dish increases the risk of polyspermy and could be detrimental for early stages of embryonic development. We identified a time window within which fertilization occurs, in order to reduce the length of sperm-egg co-incubation and optimize the procedure, comparing polyspermy rate and embryo development after short (shIVF) and overnight (o/nIVF) spermatozoa-oocyte co-incubation. A total of 666 invitro-matured sheep oocytes were co-incubated with spermatozoa in IVF medium (synthetic oviductal fluid (SOF) with 20% oestrus sheep serum and 16 µM isoproterenol). First, small batches of oocytes were collected every 30min to check for the presence of a fertilizing spermatozoon. To assess this, cumulus cells were removed and presumptive fertilized oocytes were fixed and stained with propidium iodide for nuclei and Pisum sativum agglutinin for zona pellucida (ZP) detection, respectively. Then, pronuclear formation (PN) and embryo development were evaluated after 16h (PN), 24h (2 cells), and 7 days of culture (blastocyst). The oocytes that were not cleaved at 24h were stained for DNA content with Hoechst 33342. Furthermore, we evaluated embryo quality by counting cells of 8-day blastocysts after differential staining of inner cell mass (ICM) and trophectoderm (TE). We found that spermatozoa reach the ZP no earlier than 90min from the beginning of co-incubation and achieve fertilization within 4h. Polyspermic fertilization (&amp;gt;2PN) was lower in shIVF (6.5%) than in o/nIVF (17.8%; P=0.006). This proportion of polyspermy was maintained between groups in noncleaved oocytes at 24h from fertilization. Likewise, cleavage and blastocyst rate were higher in shIVF compared with the o/n-IVF group (2-cells: 48.3% vs. 31.6%, P=0.001; blastocyst: 29.4% vs. 20.5%, P=0.046, respectively). Differential staining of blastocysts revealed no significant difference in cell number between the blastocysts of the two groups. This work demonstrates that 4h of sperm-egg interaction are sufficient to achieve fertilization, reduce polyspermy, and improve the rate of embryos reaching blastocyst stage without compromising embryo quality.

Pleiotropic effects of alpha-SNAP M105I mutation on oocyte biology: ultrastructural and cellular changes that adversely affect female fertility in mice

After sperm-oocyte fusion, cortical granules (CGs) located in oocyte cortex undergo exocytosis and their content is released into the perivitelline space to avoid polyspermy. Thus, cortical granule exocytosis (CGE) is a key process for fertilization success. We have demonstrated that alpha-SNAP -and its functional partner NSF- mediate fusion of CGs with the plasma membrane in mouse oocytes. Here, we examined at cellular and ultrastructural level oocytes from hyh (hydrocephalus with hop gait) mice, which present a missense mutation in the Napa gene that results in the substitution of methionine for isoleucine at position 105 (M105I) of alpha-SNAP. Mutated alpha-SNAP was mislocalized in hyh oocytes while NSF expression increased during oocyte maturation. Staining of CGs showed that 9.8% of hyh oocytes had abnormal localization of CGs and oval shape. Functional tests showed that CGE was impaired in hyh oocytes. Interestingly, in vitro fertilization assays showed a decreased fertilization rate for hyh oocytes. Furthermore, fertilized hyh oocytes presented an increased polyspermy rate compared to wild type ones. At ultrastructural level, hyh oocytes showed small mitochondria and a striking accumulation and secretion of degradative structures. Our findings demonstrate the negative effects of alpha-SNAP M105 mutation on oocyte biology and further confirm the relevance of alpha-SNAP in female fertility.

Exposure to Waterborne nTiO2 Reduces Fertilization Success and Increases Polyspermy in a Bivalve Mollusc: A Threat to Population Recruitment.

Fertilization success is crucial for the population recruitment of an organism. However, little is known about the threat of nanoparticles (NPs) to the fertilization of broadcast spawners. Therefore, the effects of nTiO2 on fertilization success, polyspermy rate, sperm velocity, gametic DNA damage, sperm-egg collision probability, gamete fusion, and oocyte ultrastructure were investigated in a broadcast spawning bivalve, Tegillarca granosa. The results obtained show that fertilization success significantly decreased, whereas polyspermy risk markedly increased upon nTiO2 exposure. In addition, nTiO2 exposure led to a significant reduction in sperm swimming velocity, which would subsequently constrain gamete collisions. In addition, nTiO2 exposure resulted in a significant decline in gamete fusion per collision along with aggravated DNA damage in gametes. Furthermore, ultrastructural analysis illustrated the attachment of nTiO2 to the oocyte surface, which subsequently resulted in microvillus disassociation and plasma membrane damage. In conclusion, the results obtained suggest a significant threat from NP pollution to the recruitment of broadcast spawning invertebrates.

Altered actin cytoskeleton in ageing eggs of starfish affects fertilization process

Integrity of oocytes is of pivotal interest in the medical and zootechnical practice of in vitro fertilization. With time, oocytes undergo deterioration in quality, and ageing oocytes often exhibit compromised competence in fertilization and the subsequent embryonic development. With ageing oocytes and eggs of starfish (Astropecten aranciacus), we addressed the issue by examining changes of the subcellular structure and their performance at fertilization. Ageing eggs were simulated in two different experimental paradigms: i) oocytes were overmatured by 6 hours stimulation with 1-methyladenine (1-MA); ii) oocytes were removed from the gonad and maintained in seawater for 24 or 48 h before applying the hormonal stimulation (1-MA, 70 min). These eggs were compared with normally matured eggs (stimulated after isolation from the gonad with 1-MA for 70 min) with respect to the sperm-induced intracellular Ca2+ signaling and the structural changes of the egg surface. The cytoskeletal and ultrastructural differences in these eggs were assessed by confocal and transmission electron microscopy, respectively. In the two categories of ageing eggs, we have found remarkable structural modifications of the actin cytoskeleton and the cortical vesicles beneath the plasma membrane. At fertilization, these ageing eggs manifested an altered pattern of intracellular Ca2+ release, aberrant actin dynamics, and increased rate of polyspermy often despite full elevation of the fertilization envelope. Taken together, our results highlight the importance of spatio-temporal regulation of the actin cytoskeleton in the cortex of the eggs, and we postulate that the status of the actin cytoskeleton is one of the major determinants of the oocyte quality that ensures successful monospermic fertilization.

The comparison of three media on the &lt;i&gt;in vitro&lt;/i&gt; maturation rate of pig oocytes

In vitro embryo production in pigs remains a challenging obstacle, given the high rate of polyspermy following in vitro fertilization. Improving the efficiency of successful in vitro maturation, which leads to sequential in vitro fertilization and culture, augments the need for modern biotechnologies in pig farming systems, and subsequently conserves germplasm, that is, embryos to be used in future breeding programs. Thus, in this study the effects were compared of North Carolina State University-23 (NCSU-23), North Carolina State University-37 (NCSU-37), and epidermal growth factor (EGF/TCM-199) media on pig oocyte maturation rate and subsequent polar body formations in vitro . Ovaries were collected from a local abattoir in a controlled temperature of 39 °C. The oocytes were aspirated with an 18-gauge needle. Oocytes surrounded by a uniform complex-cumulus mass were selected. A total of 284 oocytes were allocated to the study, and were incubated with 5% CO 2 at 39 °C for 48 hours. In vitro maturation rate was observed by checking the presence of expanded cumulus-oocyte complex (COC) and polar body formations under a light microscope. The maturation rate and polar body formations were significantly higher in NCSU-37 with 85.9% and 81.9%, respectively. However, there were no significant differences in EGF and NCSU-23 media as 77.1%; 72.5% and 77.1%; 72.7% were obtained for maturation rate and polar body formations, respectively. In conclusion, EGF and NCSU-23 had lower rates of maturation and meiotic development owing to their dissimilarities in composition. However, NSCU-37 rendered the highest results because the composition of the media contained beneficial constituents, that is, gonadotropins. New assisted reproductive programs may be important for commercial pig farming systems, as this may facilitate innovative ways of production. Keywords : Embryo production, genetic resources, pig production, reproductive biotechnology

Superstimulation with eCG prior to ovum pick-up improves follicular development and fertilization rate of cattle oocytes

This study aimed to evaluate the effect of equine chorionic gonadotrophin (eCG) stimulation prior to ovum pick-up (OPU) on follicular development, number and quality of recovered oocytes, fertilization rate, and early embryo development in vitro. There were four OPU sessions (cross over) conducted on 16 Braford cows to evaluate the effect of various eCG doses. The timing of the wave of ovarian follicular development was synchronized, and three days after, the respective eCG dose was administrated (0, 200, 400, or 800 IU). The OPU was performed on Day 6, and viable oocytes were used for IVM and IVF according to the respective treatment. After IVF treatment, the fertilization and cleavage rates, time of cleavage, and the cell number at 48 h were evaluated. There was no difference in the number of follicles, oocyte quantity, and morphological quality of oocytes among treatments (P > 0.05). The oocyte recovery rate was similar among the eCG-treated groups, but was less than in the control group (P < 0.01). The eCG800 group, however, had a greater recovery rate of follicles >6 mm in diameter (P < 0.01). In addition, the eCG800 group had a greater rate of normal fertilization (P < 0.01) and lesser rate of polyspermy (P < 0.02). The cleavage rate of the eCG800 group was greater than the other treatment groups but similar to that of the control. In conclusion, the use of eCG800 increased the proportion of follicles > 6 mm, with improved rate of normal fertilization and reduced occurrence of polyspermy, without affecting early embryonic development in vitro.

Calcium-sensing receptor (CASR) is involved in porcine in vitro fertilisation and early embryo development.

It has been demonstrated that extracellular calcium is necessary in fertilisation and embryo development but the mechanism is still not well understood. The present study mainly focussed on the extracellular calcium effector called the calcium-sensing receptor (CASR) and examined its expression in porcine gametes and embryos and its function during fertilisation and early embryo development. By using reverse transcription polymerase chain reaction, CASR was found to be expressed in porcine oocytes, spermatozoa and embryos at different developmental stages. Functionally, medium supplementation with a CASR agonist or an antagonist during in vitro fertilisation (IVF) and in vitro culture (IVC) was tested. During fertilisation, the presence of a CASR agonist increased sperm penetration rate and decreased polyspermy rate leading to an increased normal fertilisation rate. During embryo development, for the IVF embryos, agonist treatment during IVC significantly increased cleavage rate and blastocyst formation rate compared with the control group. Furthermore, parthenogenetically activated embryos showed similar results with lower cleavage and blastocyst formation rates in the antagonist group than in the other groups. It was concluded that CASR, as the effector of extracellular calcium, modulates porcine fertilisation and early embryo development.

Polarization microscopy imaging for the identification of unfertilized oocytes after short-term insemination

To develop a unique approach using polarization microscopy (PM) to determine whether the presence of a spindle can be used as an indicator associated with fertilization failure 5hours after short-term insemination. Observational study. Assisted reproduction center. Eighty-five patients undergoing short-term insemination. Oocytes imaged via PM at 4, 5, and 6hours after standard insemination. Spindle visualization and fertilization rate, with rescue intracytoplasmic sperm injection (ICSI) results determined by rates of normal fertilization, abnormal fertilization, and good-quality embryo formation. After standard insemination, comparisons of spindle visualization at three time points indicated that the predictive accuracy rates were 84.30% at 5hours, 86.80% at 6hours, and 62.20% at 4hours, with the rates at 5 and 6hours statistically significantly higher than at 4hours. A spindle was present in 242 of the 788 metaphase-II oocytes 5hours after insemination, and there were 204 failed fertilizations on day 1. The positive predictive value was 0.84. After rescue ICSI, the abnormal fertilization rate of the polar body group (assessed using the polar body visualization method) was statistically significantly higher than that of the PM group (assessed using the spindle visualization method) and the regular ICSI group (9.37%, 5.88%, and 4.87%, respectively). The presence of a spindle 5hours after insemination in invitro fertilization is an accurate indicator of unfertilized oocytes. Spindle imaging combined with rescue measures effectively prevents fertilization failure and decreases the polyspermy rate.

In vitro fertilization of porcine oocytes is affected by spermatic coincubation time

Abstract: The aim was to study the effects of different gamete coincubation times on porcine in vitro fertilization (IVF), and to verify whether efficiency could be improved by reducing oocyte exposure time to spermatozoa during IVF. In groups of 50, a total of 508 immature cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium. The COCs were cultured for 44 hours and then inseminated with in natura semen (2,000 spermatozoa/oocyte). The sperm and oocytes were coincubated according to the following treatments (T): T1 = oocytes exposed to spermatozoa for one hour (173 oocytes), T2 = oocytes exposed to spermatozoa for two hours (170 oocytes), and T3 = oocytes exposed to spermatozoa for three hours (165 oocytes). After these coincubation periods, the oocytes were washed in fertilization medium (TALP medium) to remove spermatozoa not bound to the zona pellucida and cultured in another similar medium (containing no sperm). Eighteen to twenty hours after fertilization, the putative zygotes were stained in Hoechst-33342 to evaluate the IVF results. The penetration rate was higher (P&lt;0.05) after two hours of coincubation time than it was for one or three hours. Furthermore, 68.60% of the ova coincubated with the spermatozoa for two hours were monospermic. The oocytes exposed to spermatozoa for one hour (T1) presented a higher (P&lt;0.01) rate of polyspermy than those in T2 and T3. Fertilization performance (%) did not differ (P&gt;0.05) between oocytes exposed to spermatozoa for one (T1) and three hours (T3). However, optimum (P=0.048) results were obtained after two hours of coincubation, when the rate of fertilization performance was 50.16±8.52%. The number of penetrated sperm per oocyte, as well as male pronucleus formation, did not differ (P&gt;0.05) between the treatments evaluated. Under these assay conditions, especially in relation to the sperm concentration used, gamete coincubation for a period of two hours appears to be optimal for monospermy and fertilization performance. Thus, it is the optimal time period for obtaining a large number of pig embryos capable of normal development.

Effects of cumulus cell removal time during in vitro fertilization on embryo quality and pregnancy outcomes: a prospective randomized sibling-oocyte study.

BackgroundTo investigate whether cumulus cell removal after a 3 h co-incubation of gametes can affect the outcomes of in vitro fertilization.MethodsA prospective randomized sibling-oocyte study was performed in which sibling oocytes obtained from each patient were randomly assigned to either a 3 h or 20 h group (cumulus cells removed at 3 h or 20 h after insemination, respectively). Same origin embryos (either 3 h or 20 h) were transferred. The study participants were patients < 38 years old and with infertility due to tubal factors. The study outcomes included fertilization, embryo quality, and birth status.ResultsThe study enrolled 172 patients, from whom 2275 oocytes were retrieved (1139 oocytes for the 3 h group and 1136 oocytes for the 20 h group). A total of 134 patients received embryo transfers (74 patients in the 3 h group and 60 patients in the 20 h group), and there were 54 live births (32 in the 3 h group and 22 in the 20 h group). When compared with patients in the 20 h group, patients in the 3 h group produced a larger number of optimal quality embryos, but had higher rates of polyspermy and low birth weight newborns. The two groups showed no differences in their rates of normal fertilization, pregnancy, and live birth.ConclusionsWhen compared with results obtained using a traditional cumulus cell removal protocol, early cumulus cell removal has both advantages and disadvantages. Further studies, and especially long-term outcome studies on newborns, need to be performed.Trial registrationCurrent controlled trial ChiCTR-OOC-15006878

Effect of spermatozoa motility hyperactivation factors and gamete coincubation duration on in vitro bovine embryo development using flow cytometrically sorted spermatozoa.

The aim of the present study was to evaluate the effects of sperm motility enhancers and different IVF times on cleavage, polyspermy, blastocyst formation, embryo quality and hatching ability. In Experiment 1, sex-sorted X chromosome-bearing Bos taurus spermatozoa were incubated for 30min before 18h fertilisation with hyperactivating factors, namely 10mM caffeine (CA), 5mM theophylline (TH), 10mM caffeine and 5mM theophylline (CA+TH); and untreated spermatozoa (control). In Experiment 2, matured B. taurus oocytes were fertilised using a short (8h) or standard (18h) fertilisation length, comparing two different fertilisation media, namely synthetic oviducal fluid (SOF) fertilisation medium (SOF-FERT) and M199 fertilisation medium (M199-FERT). Cleavage and blastocyst formation rates were significantly higher in the CA+TH group (77% and 27%, respectively) compared with the control group (71% and 21%, respectively). Cleavage rates and blastocyst formation were significantly lower for the shortest fertilisation time (8h) in M199-FERT medium (42% and 12%, respectively). The SOF-FERT medium with an 8h fertilisation time resulted in the highest cleavage rates and blastocyst formation (74% and 29%, respectively). The SOF-FERT medium produced the highest embryo quality (50% Grade 1) and hatching rate (66%). Motility enhancers did not affect polyspermy rates, whereas polyspermy was affected when fertilisation length was extended from 8h (3%) to 18h (9%) and in M199-FERT (14%) compared with SOF-FERT (6%). We conclude that adding the motility enhancers CA and TH to sex sorted spermatozoa and Tyrode's albumin lactate pyruvate (TALP)-Sperm can improve cleavage and embryo development rates without increasing polyspermy. In addition, shortening the oocyte-sperm coincubation time (8h) resulted in similar overall embryo performance rates compared with the prolonged (18h) interval.

Clinical and Functional Analyses of Frozen Porcine Sperm that Indicate IVF Success

counted. We found that ejaculates containing higher numbers of sperm bound to oviduct aggregates were related to IVF polyspermy rates (r 2 =0.62). Polyspermy is common when using porcine gametes and is particularly frequent when using high quality semen samples. �怀ese results are consistent with the principle that oviduct-bound sperm are likely more functional because they have not undergone capacitation and have less cryo-induced damage and are therefore, more e�耀ective at fertilization [4-7]. In conclusion, sperm traits that are o�耀en used for clinical assessment, such as motility and acrosome integrity, were not strongly predictive of IVF success. However, a binding assay that determined the number of sperm bound to oviduct epithelial aggregates was predictive of IVF polyspermy rates. Our observations indicate that the oviduct has an important role in contributing to in vitro fertilization and support a need for the development of assays that assess sperm function as a more powerful tool to screen samples that are compatible for technologies such as IVF.