Rate Of Polyspermic Fertilization Research Articles

Dithiothreitol Affects the Fertilization Response in Immature and Maturing Starfish Oocytes.

Immature starfish oocytes isolated from the ovary are susceptible to polyspermy due to the structural organization of the vitelline layer covering the oocyte plasma membrane, as well as the distribution and biochemical properties of the actin cytoskeleton of the oocyte cortex. After the resumption of the meiotic cycle of the oocyte triggered by the hormone 1-methyladenine, the maturing oocyte reaches fertilizable conditions to be stimulated by only one sperm with a normal Ca2+ response and cortical reaction. This cytoplasmic ripening of the oocyte, resulting in normal fertilization and development, is due to the remodeling of the cortical actin cytoskeleton and germinal vesicle breakdown (GVBD). Since disulfide-reducing agents such as dithiothreitol (DTT) are known to induce the maturation and GVBD of oocytes in many species of starfish, we analyzed the pattern of the fertilization response displayed by Astropecten aranciacus oocytes pre-exposed to DTT with or without 1-MA stimulation. Short treatment of A. aranciacus immature oocytes with DTT reduced the rate of polyspermic fertilization and altered the sperm-induced Ca2+ response by changing the morphology of microvilli, cortical granules, and biochemical properties of the cortical F-actin. At variance with 1-MA, the DTT treatment of immature starfish oocytes for 70 min did not induce GVBD. On the other hand, the DTT treatment caused an alteration in microvilli morphology and a drastic depolymerization of the cortical F-actin, which impaired the sperm-induced Ca2+ response at fertilization and the subsequent embryonic development.

Linoleic and linolenic acids reduce the effects of heat stress-induced damage in pig oocytes during maturation in vitro.

Elevated environmental temperatures can induce heat stress which could reduce fertility and early embryonic development. Fatty acids can initiate an endergonic reaction that absorbs cellular heat and decreases intracellular temperature. This study's objective was to minimize heat stress-induced damage to in vitro matured oocytes by supplementing maturation media with either 50μM linoleic or linolenic acid or both (25 or 50μM) during maturation at either 38.5 or 41.5°C. Oocytes were evaluated for intracellular antioxidative pathways, fertilization characteristics, or early embryonic development. Elevated maturation temperatures increased (p < 0.05) reactive oxygen species (ROS) formation and supplementation with linoleic or linolenic acid decreased (p < 0.05) ROS in oocytes matured at 41.5°C. Maturation temperature had an effect (p < 0.05) on the intracellular antioxidative pathways of the oocyte except for glutathione peroxidase activity. Regardless of maturation temperature, supplementation with linoleic or linolenic acid increased (p < 0.05) the enzyme activities and glutathione concentrations in the oocytes compared to no fatty acid supplementation. Supplementation of both linoleic and linolenic acid decreased (p < 0.05) polyspermic fertilization rates. Supplementing either 25 or 50μM linoleic and linolenic acid to maturing oocytes at 41.5°C increased (p < 0.05) cleavage rates by 48h after IVF and the blastocyst formation rates by 144h after IVF compared to other treatments. Oocytes matured at 38.5°C had greater (p < 0.05) embryonic development than those matured at 41.5°C except for those supplemented with 50μM linoleic and linolenic acid. Supplementing 50μM linoleic and linolenic acid to the maturation medium of pig oocytes reduces the effects of heat stress-induced damage.

Removal of sialic acid from bull sperm decreases motility and mucus penetration ability but increases zona pellucida binding and polyspermic penetration in vitro.

This study tested the hypothesis that sperm sialic acid (Sia) is required to reach the site of fertilization, and that successful fertilization requires recognition of Sia from both the sperm and oocyte to occur. In addition, it has recently been reported that Siglecs (Sia-binding-immunoglobulin-like lectins) are present on the sperm surface. Thus, the possibility that the recognition of oocyte Sia was sperm-Siglec-mediated was also addressed. Sperm exposed to neuraminidase (NMase) exhibited lower overall and progressive motility, which translated to a decreased ability to swim through cervical mucus from cows in oestrus. In addition, when either sperm or cumulus-oocyte complexes (COCs) were treated with NMase, a decrease in cleavage and blastocyst rate was observed. However, incubation of sperm with increasing concentrations of anti-Siglec-2, -5, -6 and -10 antibodies prior to fertilization had no effect on their fertilizing ability. Interestingly, treatment with NMase increased the number of sperm bound to the ZP but also the rate of polyspermic fertilization. Flow cytometry analysis revealed no differences in the percentage of capacitated or acrosome-reacted sperm. These results suggest that Sia are required to reach the site of fertilization but need to be removed for sperm-oocyte interaction. However, fine regulation is needed to avoid abnormal fertilization which can lead to impaired embryo development.

Effects of Exposure to Zearalenone on Porcine Oocytes and Sperm During Maturation and Fertilization In Vitro

The influence of acute exposure to zearalenone (ZEN) on porcine oocyte maturation, fertilization or sperm penetration ability during both in vitro maturation and fertilization was evaluated. First, oocytes were cultured in ZEN-containing (0-1000 µg/l) maturation medium and then fertilized. The oocytes maturing in vitro without ZEN were then fertilized in ZEN-containing fertilization medium. The maturation rates of oocytes and penetration ability of sperm decreased significantly in the presence of 1000 µg/l of ZEN. However, neither increases in the rates of degeneration and DNA fragmentation of oocytes nor reductions in normal and polyspermic fertilization were observed. ZEN did not affect the sperm penetration rates; however, 1000 µg/l ZEN had positive effects on normal and polyspermic fertilization rates. Therefore, it can be suggested that an acute exposure of porcine oocytes during maturation and of oocytes and sperm during fertilization to ZEN up to 1000 µg/l may not affect the fertility of the oocytes.

Follicle Growth and Oocyte Developmental Competence in Cows With Liver Damage

Follicle growth, oocyte quality or oocyte growing environment (follicular fluid) were evaluated in cows with severe liver damage (haemorrhage, telangiectasis, cholangitis and abscess) that were visually diagnosed at the slaughterhouse. Holstein cows aged 40-90 months with either a healthy liver (HL cow) or damaged liver (DL cow) were selected as donors. Follicle development kinetics was evaluated by counting the follicles at various developmental stages. In addition, the biochemical characteristics of the follicular fluids, developmental competence of preantral follicles cultured for 16 days in vitro and the ability of oocytes to develop to the blastocyst stage 8 days after fertilization were examined. DL cows had fewer secondary follicles than HL cows, and the correlation between the number of secondary follicles and the number of primary follicles differed among DL and HL cows. The follicular fluid of DL cows contained significantly lower levels of albumin and a higher total protein content than that of HL cows. Oocyte nuclear maturation assessed at 5, 16 and 21 h after beginning of culture was slower in DL cows than in HL cows, although the final maturation rates did not differ. The rate of polyspermic fertilization was significantly higher and the proportion of cleavage at 48 h after insemination and blastulation lower in DL cows compared with HL cows. When preantral follicles were cultured in vitro, the rate of follicles with normal morphology was lower in DL cows than in HL cows. These findings suggest that the kinetics of folliculogenesis differ among DL and HL cows and the developmental ability of preantral follicles and oocytes is lower in DL cows than in HL cows.

Effect of N-Acetyl-D-Glucosamine on Bovine Sperm-Oocyte Interactions

N-Acetyl-D-glucosamine (GlcNAc) is a major component of glycosaminoglycan, which is involved in sperm-oocyte interactions. We examined the effect of adding GlcNAc and other monosaccharides, D-mannose and D-fucose, to the in vitro fertilization (IVF) medium on bovine sperm-oocyte interactions. In medium in which sperm and a zona pellucida (ZP) were co-incubated with monosaccharides for 5 min, addition of GlcNAc (5 or 25 mM) significantly reduced the number of sperm that attached to the ZP. Pretreatment of gametes with GlcNAc (5 mM) prior to co-incubation also suppressed sperm-ZP attachment. Addition of GlcNAc (5 or 25 mM) to the medium in which sperm and a ZP were co-incubated for 5 h, however, significantly increased the number of sperm binding to and penetrating the ZP in a concentration-related manner. The other monosaccharides, D-fucose and D-mannose, did not have this effect. Supplementation of the sperm-oocyte co-incubation medium with 5 mM GlcNAc also enhanced the rate of polyspermic fertilization. When the ZPs were removed from the oocytes, GlcNAc did not affect the fertilization rate. Furthermore, incubation of sperm with 5 mM GlcNAc induced sperm membrane destabilization and an acrosome reaction, as evidenced by the hypo-osmotic swelling test and fluorescein isothiocyanate-labeled peanut agglutinin/propidium iodide (FITC-PNA/PI) staining. Finally, GlcNAc suppressed ZP hardening following fertilization, as determined by measuring the time required for pronase to dissolve the ZP. In conclusion, supplementation of IVF medium with GlcNAc has various effects on sperm-oocyte interactions including suppression of initial attachment, induction of sperm membrane destabilization and acrosome reaction, increase in the number of sperm secondarily bound to and penetrating the ZP, suppression of ZP hardening following sperm-oocyte co-incubation and increase in the rate of polyspermic fertilization.

Effect of N-acetyl-D-glucosamine on Bovine Sperm-oocyte Interactions.

N-Acetyl-D-glucosamine (GlcNAc) is a major component of glycosaminoglycan, which is involved in sperm-oocyte interactions. We examined the effect of adding GlcNAc and other monosaccharides, D-mannose and D-fucose, to the in vitro fertilization (IVF) medium on bovine sperm-oocyte interactions. In medium in which sperm and a zona pellucida (ZP) were co-incubated with monosaccharides for 5 min, addition of GlcNAc (5 or 25 mM) significantly reduced the number of sperm that attached to the ZP. Pretreatment of gametes with GlcNAc (5 mM) prior to co-incubation also suppressed sperm-ZP attachment. Addition of GlcNAc (5 or 25 mM) to the medium in which sperm and a ZP were co-incubated for 5 h, however, significantly increased the number of sperm binding to and penetrating the ZP in a concentration-related manner. The other monosaccharides, D-fucose and D-mannose, did not have this effect. Supplementation of the sperm-oocyte co-incubation medium with 5 mM GlcNAc also enhanced the rate of polyspermic fertilization. When the ZPs were removed from the oocytes, GlcNAc did not affect the fertilization rate. Furthermore, incubation of sperm with 5 mM GlcNAc induced sperm membrane destabilization and an acrosome reaction, as evidenced by the hypo-osmotic swelling test and fluorescein isothiocyanate-labeled peanut agglutinin/propidium iodide (FITC-PNA/PI) staining. Finally, GlcNAc suppressed ZP hardening following fertilization, as determined by measuring the time required for pronase to dissolve the ZP. In conclusion, supplementation of IVF medium with GlcNAc has various effects on sperm-oocyte interactions including suppression of initial attachment, induction of sperm membrane destabilization and acrosome reaction, increase in the number of sperm secondarily bound to and penetrating the ZP, suppression of ZP hardening following sperm-oocyte co-incubation and increase in the rate of polyspermic fertilization.

Dielectrophoretic oocyte selection chip for in vitro fertilization

This paper reports a new dielectrophoretic separation method of porcine oocytes for in vitro fertilization. Conventional manual selection of oocyte highly depends on the expert's experience and lacks universal standards for identifying the quality of oocyte. In this study, an electrode array chip with castellated shape was developed to evaluate dielectrophoretic velocities of oocytes, under applied bias conditions with an AC 3 V waveform at 1 MHz for 15 s. Based on different dielectrophoresis (DEP) response, the selected group of oocytes that moved showed a better developmental potential than the group of oocytes that stayed, representing a higher rate of blastocyst formation and a lower rate of polyspermic fertilization. In addition, the overall developmental potential of oocytes selected by the DEP device was comparable to that of oocytes selected by conventional manual method. These results demonstrate that the difference in dielectrophoretic velocity can be used to establish an objective criterion for the selection of oocytes. Consequently, this method will open the possibility to develop an automatic tool for oocyte selection, which would be helpful for assisted reproductive technologies such as transgenic and clonal animal production.

Inhibitory effects of calcium ionophore pretreatment of porcine oocytes on polyspermic fertilization

The present study examined the inhibitory effects of various pretreatment concentrations (0-100 microM) of the calcium ionophore A23187 on polyspermic fertilization and then examined the effect of the maturation period and the time between calcium ionophore treatment and fertilization on the inhibitory effect of calcium ionophore on polyspermic fertilization. In experiment 1, a high concentration of calcium ionophore (100 microM) increased the rate of activated oocytes, but the rate of fertilization declined. On the other hand, when oocytes were treated with a low concentration of calcium ionophore (10 microM), monospermic fertilization was significantly increased (10 microM; 31.3%) (p < 0.05). In experiment 2, oocytes were cultured for various times (0, 0.5, 3, 6 h) after calcium ionophore treatment (10 microM) before fertilization. The highest rate of monospermic fertilization was detected in the oocytes cultured for 6 h after calcium ionophore treatment before fertilization. In experiments 3 and 4, we examined the effect of the maturation period (40 h or 44 h) on the rate of fertilization and blastulation of oocytes pretreated with calcium ionophore. The treatment of oocytes with calcium ionophore significantly decreased the rate of polyspermic fertilization regardless of the maturation period (44 h: with calcium ionophore 26.25% vs without 78.8%; 40 h: with calcium ionophore 37.5% vs without 77.5%); however, calcium ionophore treatment increased the rates of monospermic fertilization and blastulation of the oocytes matured for 44 h, but not those matured for 40 h. In conclusion, activation with a low concentration of calcium ionophore (10 microM) and a further 6 h of culture before fertilization improved the rate of monospermic fertilization and blastulation.

312 EXPOSURE OF SPERMATOZOA TO SOLUBILIZED EXTRACTS OF THE OVIDUCTAL EPITHELIUM APICAL PLASMA MEMBRANE ENHANCES FERTILIZATION IN PORCINE IN VITRO FERTILIZATION

In vitro production (IVP) of porcine embryos is currently suboptimal compared with IVP in species such as mice and cattle. In vitro fertilization (IVF) usually involves the co-culture of oocytes and spermatozoa in a medium droplet. Oocyte quality is the focus of many studies. In vivo, the quality of spermatozoa is as important as the oocyte, and females have many mechanisms to select the highest quality spermatozoa for their oocytes. Oviductal proteins have been shown to affect sperm motility of subpopulations within an ejaculate. The present study was carried out to investigate normal and polyspermic fertilization rates of spermatozoa exposed to oviductal epithelial apical plasma membrane (APM) proteins, a mixture of peripheral proteins extracted by 1 M NaCl from isolated oviductal apical plasma membranes, prior to co-culture with oocytes in IVF. Porcine oocytes were aspirated from ovaries and grade I quality oocytes (cumulus–oocyte complexes with a spherical shape, visible nucleus, even-density cytoplasm, and multiple layers of cumulus cells) were selected and matured for 48 h in TCM-199 supplemented with LH (0.5 �g mL-1), FSH (0.5 �g mL-1), and EGF (10 ng mL-1). Ejaculates were washed through a Percoll gradient to obtain a concentrated pellet. Spermatozoa were diluted in capacitation–fertilization medium in the presence or absence of APM proteins (100 �g mL-1), incubated for 10 min, and then co-cultured with oocytes for 6 h in modified Tween medium B with milk powder medium (Abeydeera and Day 1997 Theriogenology 48, 537–544) supplemented with BSA (0.4%) and sodium bicarbonate (5 mM). Presumptive zygotes were cultured in NCSU23 medium for a further 48 h. The oocytes/zygotes were then fixed and stained with propidium iodide for evaluation by confocal microscopy for fertilization and cleavage (n = 1235 oocytes). Fertilization rates were compared between treatments in a chi-squared test using the Mantel-Haenszel approach. The overall fertilization rate was significantly higher (78 vs. 86%) when spermatozoa were incubated in the presence of APM proteins (P &amp;lt; 0.05), and in the group of fertilized oocytes, polyspermic fertilization (47 vs. 21%) was significantly reduced when spermatozoa were exposed to APM proteins (P &amp;lt; 0.01). However, cleavage rates were not different. These results suggest that exposure of spermatozoa to APM proteins prior to IVF increases the fertilization rate and decreases the incidence of polyspermic penetration.

Effect of sperm survival and CTC staining pattern on in vitro fertilization of porcine oocytes

Polyspermy in pig oocytes fertilized in vitro remains unacceptably high. In this study, we evaluated the effects of gamete coincubation time, and determined if the proportion of capacitated spermatozoa would be predictive of the fertilizing ability of frozen-thawed semen in vitro. Cumulus–oocyte complexes were collected from slaughterhouse prepubertal gilt ovaries and matured in vitro for 44 h in TCM199, with EGF, FSH, cysteamine and follicular fluid. Fertilization was induced with 2×10 5 frozen-thawed spermatozoa/ml in TBM. Penetration of oocytes as well as polyspermic fertilization occurred 2 h after insemination. A strong correlation between penetration and polyspermic fertilization rates has been demonstrated, but there was no correlation between the proportion of capacitated spermatozoa, as assessed by chlortetracycline staining, at the time of insemination and fertilization rates. We also compared the results of IVF in three IVF media: TBM, m199 and TALP. Penetration and polyspermy were very different in these three media: 71±19% and 25±13% in TBM, 37±11% and 6±2% in m199, 10±2% and 0% in TALP, respectively. Nevertheless, survival of spermatozoa or modifications of the capacitation status were not different in these media after 6 h incubation. We concluded that survival and capacitation characteristics of the semen used for IVF could not be predictive of the IVF results. It seems necessary to act at the oocyte level to control both variability between replicates and the incidence of polyspermy. Improving the spermatozoa penetration blocking system of the oocytes and reducing the number of sperm-binding sites on the zona pellucida (ZP) are our further objectives.

Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes.

Effects of adenosine and pGlu-Glu-ProNH(2) (FPP) on the function and in vitro penetration of boar spermatozoa were examined. First, the effects of dibutyryl cAMP or agonists and antagonists of adenosine receptors (inhibitory adenosine receptors, A1AdR; stimulatory adenosine receptors, A2AdR) on freshly ejaculated spermatozoa were determined by chlortetracycline fluorescence assessment. Capacitation of spermatozoa was stimulated when they were cultured in a medium with dibutyryl cAMP, adenosine, A2AdR agonist, and adenosine plus A1AdR antagonist (CPT). However, acrosome reaction was inhibited only by adenosine. A1AdR agonist did not affect intact spermatozoa. A2AdR antagonist (DMPX) neutralized all of the effects of adenosine. Second, interaction of adenosine and FPP was examined. Gln-FPP, a competitive inhibitor of FPP, and DMPX inhibited the effects of adenosine and FPP, and CPT neutralized the inhibitory effect of FPP on acrosome reaction. Last, the effects of adenosine, FPP, and caffeine on the rate of sperm penetration were examined using frozen-thawed spermatozoa. Adenosine, FPP, and caffeine significantly enhanced the rate of sperm penetration as compared with the case of no additions. Caffeine treatment resulted in a high rate of polyspermic fertilization. In contrast, adenosine and FPP treatments resulted in an increased proportion of normal fertilization in in vitro-matured oocytes. These results suggest that boar spermatozoa can be modulated by the adenylyl cyclase/cAMP pathway via A2AdR in intact cells to induce capacitation and A1AdR in capacitated cells to inhibit spontaneous acrosome loss and that FPP receptors interact with A2AdR in intact cells and with A1AdR in capacitated cells. Furthermore, adenosine and FPP seem to be useful in reducing the incidence of polyspermic penetration.

In vitro production of sexed embryos for gender preselection: high-speed sorting of X-chromosome-bearing sperm to produce pigs after embryo transfer.

The objectives for the present experiments were to apply sperm sexing technology to an in vitro production system with porcine oocytes obtained from slaughterhouse material. On six experimental days, ovaries were obtained from an abattoir, and cumulus-oocyte-complexes were matured in vitro. Semen was collected from mature boars of proven fertility and was sorted for X-chromosome-bearing sperm, using the Beltsville Sperm Sexing Technology incorporating the use of high-speed sorting. A total of 5,378 oocytes were submitted for in vitro fertilization (IVF). Of these, 559 ova were stained for cytogenetic analysis 18 h after IVF. From the remaining 4,819 ova, 1,595 cleaved, and 1,300 of the cleaved embryos were transferred into 26 synchronized recipients (5 control gilts for unsorted sperm, 21 gilts for X-sorted sperm). In a test of two fertilization media (FERT-A vs FERT-B) higher cleavage rates (P<.05) were obtained when FERT-B was used as a fertilization medium for unsorted (43.4+/-5.1%) and sorted sperm (43.1+/-1.1%;), whereas in FERT-A unsorted sperm gave a cleavage rate of 17.9+/-4.4% and sorted sperm gave 30.4+/-1.4%. Additionally, cleavage rates were higher (P<.05) after fertilization with sorted sperm vs unsorted sperm, independent of fertilization medium. Cytogenetic analysis of ova revealed that more oocytes with unsorted than with sorted sperm remained in Metaphase 2 arrest (P<.05). This was also independent of the fertilization medium. Monospermic fertilization rates were the same for IVF with unsorted or sorted sperm, independent of the fertilization system, except FERT-A with unsorted sperm (P<.05). Polyspermic fertilization rates were highest in FERT-B (37.6+/-6.6). A total of 57 pigs were born from nine litters. Six litters from sexed sperm (X-sorted) produced 33 females (97%) and one male. Three litters from control transfers produced 23 pigs, 11 of which were female (48%). The sex ratio of the offspring was predicted based on the sort reanalysis of the sorted sperm for DNA content.

Bovine oocyte diameter in relation to developmental competence

This study was conducted to determine the diameter of bovine oocytes that were able to attain their full developmental competence to blastocysts. Oocytes were recovered by aspiration of surface-visible follicles (1 to 7 mm in diameter) from slaughterhouse ovaries. Only healthy-looking cumulus-oocyte complexes were used for in vitro maturation, and they were divided into six groups based on diameter: < 110 μm, 110 to < 115 μm, 115 to < 120 μm, 120 to < 125 μm, 125 to < 130 μm and ≥ 130 μm. Oocytes were processed through standard procedures for in vitro maturation, fertilization and culture. Following in vitro maturation or fertilization, some oocytes were stained to assess nuclear maturation and penetration rates. The numbers of embryos that cleaved at 42 h post insemination and developed to blastocysts and hatched blastocysts after 8 days of culture were recorded. The mean oocyte diameters were 114.0 ± 4.8 μm. The oocytes displayed size-related ability to undergo meiotic maturation. The rates of nuclear maturation of oocytes in the greater than 115-μm size range were significantly higher than those of oocytes with diameters < 115 μm. In the < 120 μm diameter groups, the polyspermic fertilization rates of oocytes < 115 μm were significantly higher than those of oocytes 115 to < 120 μm in diameter. The rates of cleavage and development to blastocysts and hatched blastocysts rose as oocyte diameter increased. Among oocytes with a diameter >110 μm, oocytes < 120 μm were found to have significantly lower developmental competence than oocytes 120 to < 130 μm in diameter. These results suggest that bovine oocytes have acquired full meiotic competence at a diameter of 115 μm but not yet attained full developmental competence to blastocysts, and that oocytes have acquired full developmental competence at a diameter of 120 μm.

In Vitro Fertilizing Ability of Bovine Oocytes Frozen-Thawed at Immature, Maturing, and Mature Stages.

The effect of the nuclear stage during in vitro maturation (IVM) on the fertilizing ability of frozen-thawed bovine oocytes was investigated. Oocytes with compact cumulus cells were cultured for 0, 6, 12 and 24 h, and were subjected to 2-step freezing in the presence of 10% ethylene glycol and 0.1 M sucrose. After thawing, the oocytes were additionally cultured to give 24 h for a total IVM period, and were inseminated with 3.5 to 5.0 × 106 cells/ml frozen-thawed spermatozoa for 20 h in TALP solution containing 20 μg/ml heparin. In vitro fertilization rates of oocytes frozen-thawed at 0, 6, 12 and 24 h of IVM were 40, 47, 49 and 54%, respectively, all of which were significantly lower than that of nonfrozen control oocytes (77%). A higher frequency of polyspermic fertilization was observed in the oocytes frozen-thawed at 24 h of IVM (53 vs. 18% in nonfrozen control oocytes). Lowering the sperm concentration during insemination from 5.0 × 106 cells/ml to 1.0 × 105 cells/ml did not decrease the rate of polyspermic fertilization in the frozen-thawed oocytes.

Asynchronous meiotic progression in porcine oocytes matured in vitro: a cause of polyspermic fertilization?

The progression of meiosis in porcine oocytes matured in vitro was examined and the effect of maturation interval on the incidence of polyspermic fertilization and embryonic development in vitro was investigated. In Experiment 1, it was found that oocytes selected for in vitro maturation varied considerably in terms of their meiotic progression. Approximately half of the oocytes were already undergoing germinal vesicle breakdown at the start of maturation while the remainder were at the intact germinal vesicle stage. These two populations of oocytes developed to the metaphase-II stage by 24 h and 36 h of maturation respectively. Maturation for 40 h and 44 h did not increase the percentage of oocytes at metaphase-II stage observed at 36 h. In Experiment 2, reducing the maturation interval from 44 h to 36 h did not affect the percentage of oocytes penetrated but did decrease the rate of polyspermic fertilization (5% v. 34%; P < 0.05). In Experiment 3, development of the embryos produced in vitro was assessed after culture for seven days. Reducing the maturation interval from 44 h to 36 h decreased the percentage of embryos developing to the 8-cell (9% v. 18%; P < 0.05), morula (3% v. 10%; P < 0.01), and blastocyst (1% v. 8%; P < 0.001) stages. These results suggest that maturation for 44 h gives rise to a population of 'aged' oocytes which is susceptible to polyspermic fertilization.

A randomized clinical trial to evaluate the clinical effects of split- versus single-dose human menopausal gonadotropins in an assisted reproductive technology program

To compare the effect of twice (split) versus once (single) daily administration of hMG on assisted reproductive technology (ART) cycle parameters. A randomized clinical trial. Hospital-based academic ART program. A total of 171 patients undergoing their initial ART cycle were enrolled. All patients received one of two hMG regimens and were excluded from the study if they were receiving medications other than leuprolide acetate (LA) and hMG for ovulation induction. Subjects were randomized to receive an initial dose of 300 IU/d of hMG in either split-dose (i.e., 150 IU twice daily) or single-dose fashion for four successive days after down-regulation with LA. Thereafter, their daily dose was individualized, maintaining a split-or single-dose schedule. All cycles were managed in accordance with our standard ART protocols. Cancellation rate, total hMG requirements, number of days treated with hMG, E2 and P responses, oocyte yield and maturity, fertilization rate, total number of embryos, embryo quality, number of embryos transferred, implantation rate, clinical and ongoing-delivered pregnancy rates. Split-dose hMG administration resulted in a significantly higher implantation rate, but significantly lower normal and polyspermic fertilization rates than single-dose hMG administration. No significant differences were noted between the two dosage protocols with respect to the other outcome measures. Split-dose hMG administration may be associated with significantly higher implantation rates; single-dose hMG with significantly higher fertilization rates. Although our data also demonstrate a trend toward higher clinical and ongoing-delivered pregnancy rates with split-dose therapy, demonstration of a significant difference would require a multicenter trial. Based on our data, clinicians may want to consider split-dose therapy for patients with repeated implantation failures.

Chromatin and microtubule morphology during the first cell cycle in bovine zygotes

Chromatin and microtubule configurations during the first cell cycle of bovine zygotes were analyzed by DNA staining and microtubule immunolocalization using an IVM/IVF system and oocytes matured and fertilized in vivo, in order to investigate the origin of the active centrosome and to characterize the nuclear and the cytoplasmic changes following bovine fertilization. Our results suggest that the paternal centrosome is active during early zygotic development, forming a conspicuous sperm aster soon after fertilization. We also report that polyspermy in bovine eggs, leads to the formation of numerous sperm asters with different degrees of association with the chromatin. The maternal structures in both monospermic and polyspermic zygotes can be lost or degenerate. Consequently, these cells may resume the first cell cycle as androgenotes, very often with several types of mitotic activity taking place in different regions of the cell cytoplasm at the same time. As indicated by a comparison of monospermic and polyspermic fertilization rates to rates of development, it is possible that some androgenetic embryos cleave and develop to the blastocyst stage.

Presence of sperm in the perivitelline space predicts fertilization rate after partial zona dissection

To examine the association of the number of spermatozoa present in the perivitelline space and sperm parameters with fertilization after partial zona dissection in male factor patients. Partial zona dissection was applied in 62 couples (84 cycles). A total of 524 oocytes underwent partial zona dissection (1/8 of the zona circumference) (partial zona dissection group) and 171 sibling oocytes were not manipulated (control group). A total of 326 manipulated oocytes were examined for the presence of spermatozoa in the perivitelline space. University-based in vitro fertilization (IVF) program. Fifty-four (87%) couples had at least one complete failure of fertilization, and 8 (13%) couples had low fertilization rate (< 10%) in previous routine IVF attempts. Fertilization rate, cleavage rate, and the number of spermatozoa present in the perivitelline space after partial zona dissection. Monospermic and polyspermic fertilization rates were 22.3% and 6.7% in the partial zona dissection oocytes and 8.8% and 0.6% in the nonmanipulated oocytes, respectively. The cleavage rate was similar in the partial zona dissection and control group (69.2% and 66.6%, respectively). A total of 81 partial zona dissection embryos and 10 nonmanipulated embryos were transferred to the uterus of 34 women (39 cycles), resulting in four pregnancies. In 46% (18 of 39) of the patients who had both partial zona dissection and control oocytes, only the manipulated oocytes fertilized. In only 48.8% of partial zona dissection oocytes, spermatozoa were detected in the perivitelline space; in this group of oocytes the fertilization rate was 56.6%. Sperm count and morphology were not clearly correlated with the outcome of partial zona dissection. The partial zona dissection technique enhances fertilization of subfertile sperm. However, the low efficiency of the procedure, apart from being associated with a high polyspermic rate, is related to the failure of sperm to traverse the slit in the zona pellucida in approximately one half of the manipulated oocytes.

Laparoscopic and transvaginal ova recovery: the effect on ova quality

Transvaginal follicle aspiration guided by transvaginal ultrasound for ova recovery is rapidly gaining popularity in many centers practicing in vitro fertilization and embryo transfer (IVF-ET). Cycle outcome following this new method has not been directly compared to the traditional, laparoscopic recovery technique. To this end, the authors evaluated multiple parameters in 66 laparoscopic (group A), and 44 transvaginal ova recovery procedures (group B) in patients undergoing IVF-ET. No statistically significant differences could be demonstrated between the groups in all but the rate of ova fertilization. The rate of fertilization was higher in the ova recovered by transvaginal follicle aspiration (59.6 versus 69.2%; P less than 0.01). No difference could be demonstrated between the groups in the other parameters examined, which included the number of ova recovered (5.7 +/- 0.4 versus 6.0 +/- 0.7), ova maturity (87 versus 84% intermediate ova), rate of polyspermic fertilization (3.9 versus 5%), rate of cleavage (88 versus 91%), cleavage stage at transfer (3.7 +/- 0.8 versus 3.5 +/- 0.4 cells per embryo), number of embryos transferred per patient (2.7 +/- 0.1 versus 3.3 +/- 0.2), and pregnancy rates. The potential detrimental effects of general anesthesia and CO2 pneumoperitoneum present during laparoscopy but not ultrasound guided recovery on ova quality may underlie the observed difference in fertilization between the groups.