Impact of phase transition on the mouse oocyte spindle during vitrification.

During vitrification, the glass-like solidification is the phase-transition process from liquid to solid. Phase transition is one of the major factors suspected to affect the physiology of the oocyte, such as the structure of the meiotic spindle. Therefore, it is very important to investigate the systematic and morphological alterations of the metaphase-II spindle and chromosome arrangement during complete course of a vitrification and warming process. B6D2F1 (C57BL/6 X DBA/2) mouse oocytes were cryopreserved by minimum volume cooling (MVC) method of vitrification in a solution with 15% ethylene glycol, 15% dimethylsulphoxide and 0.5 mol/l sucrose. To examine the spindle, oocytes were fixed before, during and after vitrification and were analysed by immunocytochemistry and confocal microscopy. It was shown that spindles in all oocytes could be maintained through the vitrification and warming process, even though they were exposed to extreme temperature and two rounds of phase transition. According to the sequential observations, chromosome alignment was maintained throughout the complete course of vitrification, warming and post-warming stage. The impact of phase transition was barely detectable when the oocyte was exposed to the vitrification and warming process. The oocyte spindle was able to recover immediately after warming.

The oocyte spindle is preserved by 1,2-propanediol during slow freezing.

OBJECTIVE: To investigate the specific changes in oocyte spindle subjected to severe challenges of low temperature, as well as to examine the effect of cryoprotectants in preserving oocyte spindle during cryopreservation. DESIGN: In vitro experimental study. SETTING: Academic research laboratory. ANIMAL(S): B6D2F1 (C57BL/6 X DBA/2) mice. INTERVENTION(S): Mouse oocytes were cryopreserved using a slow freezing method in a sodium-depleted medium with 1.5 mol/l 1,2-propanediol (PROH) and 0.3 M sucrose. To examine the spindle, oocytes were fixed before, during, and after cryopreservation, and oocytes were analyzed by immunocytochemistry and confocal microscopy. RESULT(S): The MII spindle was preserved during the slow freezing, because the cryoprotectant PROH was found to support the organization of MII spindle in resisting the subzero temperature. In contrast, the MII spindle was disassembled gradually during the thawing process with or without PROH. Most of the oocytes were able to recover the MII spindle after thawing, but a portion of thawed oocytes could not sustain the meiotic spindle because of parthenogenetic activation. CONCLUSION(S): 1,2-Propanediol can support the organization of MII spindle to defy the subphysiologic temperature; however, the PROH cannot sustain oocyte spindle structure after the subsequent thawing process.

The efficacy and safety of human oocyte vitrification.

Vitrification is now a widely applied and highly successful approach for cryopreservation in reproductive biology. Rapidly increasing data prove that it is also a highly efficient technique for low-temperature storage of human oocytes. The latest approaches with appropriately selected cryoprotectants, tools and techniques, and properly adjusted parameters allow close to 100% morphological survival rates, and in vitro embryo development, as well pregnancy and implantation rates, comparable with those achieved with fresh oocytes. With standardization of the technique and elimination of biosafety problems by preserving all the positive features, vitrification may become a common part of the everyday routine in a human embryo laboratory, and it may offer a solution for various medical and social situations as well as for simple logistic problems commonly occurring in assisted reproduction.

Clinical evaluation of the efficiency of an oocyte donation program using egg cryo-banking.

OBJECTIVE: To evaluate the efficiency of oocyte donation cycles using egg "cryo-banking." DESIGN: Study conditions for vitrified/warmed oocytes for 20 non-autologous recipients (from 10 donors) were set prospectively, and outcomes of it were later compared retrospectively to nine fresh donations cycles. SETTING: Private assisted reproductive technology program. PATIENT(S): Ten donors and 20 infertile recipients. INTERVENTION(S): Oocytes were vitrified 3 to 4 hours after collection and cryo-stored. Intracytoplasmic sperm injection was performed 3 hours after warming, and embryos were in vitro cultured for 5 days. Two or three blastocysts were transferred per patient. MAIN OUTCOME MEASURE(S): Oocyte survival, fertilization, development, clinical pregnancy, and implantation rates. RESULT(S): A total of 153 oocytes were warmed and 134 survived. A total of 117 fertilized and 68% developed to blastocyst stage. A total of 47 embryos were transferred (2.35 embryos per recipient) and 26 implanted. Fifteen patients achieved ongoing pregnancies initially, and two additional pregnancies were obtained after transfer of supernumerary vitrified/warmed embryos. Nine of the 10 donors from the current study had previous fresh donations cycles from where seven clinical pregnancies were established in nine recipients, providing the base for comparison. CONCLUSION(S): Oocyte donation using vitrified/warmed oocytes can provide high pregnancy and implantation rates, and thus can be considered as efficient treatment procedure with additional benefits to recipients.

Human oocyte vitrification: in-vivo and in-vitro maturation outcomes.

This study aimed to evaluate oocyte vitrification efficiency using in-vivo matured (IVO) versus rescued in-vitro matured (IVM) oocytes. The results show that oocyte survival (85% versus 81%), fertilization (86% versus 76%) and cleavage rate (98% versus 89%) was not significantly different in IVO oocytes compared with rescued IVM sibling oocytes. The fertilized oocytes from IVO and IVM groups were cultured to blastocyst stage; however, embryo development was significantly reduced in the rescued IVM group (72% versus 15%). Embryo transfer was only performed with the embryos derived from IVO oocytes on day 5; 42 blastocysts were transferred to 18 recipients; 16 of 18 recipients had positive beta-human chorionic gonadotrophin and a total of 26 fetal cardiac activities were detected in 15 recipients (implantation: 26/42, 61.9%). Ten of the 15 recipients have delivered 19 healthy babies, and the other five pregnancies are still ongoing. These data indicate that the combination of oocyte vitrification and rescued IVM not only yield a new strategy to extend the pool of total fertilizable oocytes, but also demonstrate that the efficiency of vitrified/warmed oocytes can be comparable to fresh oocytes with regard to clinical outcomes.

Comparison of ICSI and conventional IVF in patients with increased oocyte immaturity.

Using sibling oocytes, the objective of this study was to compare the intracytoplasmic sperm injection (ICSI) fertilization rates to those achieved with conventional IVF in patients with high rates of oocyte immaturity. This study was observational in nature, and included 91 patients who were treated using split insemination techniques. The fertilization rates for the ICSI group and the IVF group were 41.1 +/- 15.0% and 53.2 +/- 19.8%, respectively (P <: 0.0001). There was no significant difference in day-3 embryo quality between the two groups. There was a significantly higher number of embryos frozen in the IVF group than in the ICSI group: 357 (84.8%) and 297 (76.7%), respectively (P = 0.037). Furthermore, the number of embryos either transferred or frozen was significantly higher in the IVF group than the ICSI group: 459 of 1173 (39.1%) and 385 of 1268 (30.4%), respectively (P < 0.0001). These data indicate that conventional IVF results in a higher fertilization rate than ICSI. Furthermore, IVF provided more embryos available for transfer or cryopreservation when compared with ICSI, thereby optimizing the patient's cycle.

Two successful pregnancies obtained following oocyte vitrification and embryo re-vitrification.

Recent clinical reports not only show that cryopreserved embryos can be successfully used for human fertility treatment, but also that cryopreserved oocytes may be used successfully as an adjunct to human assisted reproductive technologies. Vitrification is known to establish a glass-like solid state during the cooling process. The high concentration of cryoprotectants and an extremely rapid rate of cooling are responsible for the formation of the solid state, and also prevent formation of intracellular ice crystals. Hence, in theory, vitrification should minimize cryo-injuries, and therefore has great promise for oocyte and embryo cryopreservation. This article describes two pregnancies from vitrified-warmed blastocysts obtained after intracytoplasmic sperm injection fertilization of vitrified-warmed oocytes. Vitrification was employed to cryopreserve the oocytes and the subsequent blastocysts. The results present the intriguing implication that vitrification may serve as an efficient method for clinical oocyte cryopreservation and embryo re-cryopreservation.

Lysed cell removal promotes frozen-thawed embryo development.

OBJECTIVE: To develop a mouse model to investigate the possible causes for increased success rates when lysed cells are removed from thawed embryos. DESIGN: Experimental study. SETTING: Clinical IVF laboratory. INTERVENTION(S): Assisted hatching, cell lysis, and removal of lysed cells. MAIN OUTCOME MEASURE(S): Embryonic growth rate and morphology. RESULT(S): The mouse embryos were divided into three groups; control (no cell lysis), group 1 (cell lysis and removal), and group 2 (cell lysis only). There was no significant difference in the initial number of blastomeres in each group or the number of cells lysed artificially in groups 1 and 2. The rate of embryonic development showed a significant delay in group 2 (7.97 +/- 4.92; control, 10.42 +/- 8.18; group 1, 5.74 +/-4.42; group 2). The embryo morphology on day 4 was significantly improved in group 1 and the control group when compared with group 2. CONCLUSION(S): Mouse embryos with artificially lysed cells after thawing had poorer developmental quality and growth rates compared with control embryos. However, removal of lysed cells restored the embryo's developmental potential to that of the control. Cell number and morphology was also significantly improved compared with embryos without lysed cell removal. These findings are consistent with human embryo development after thawing when lysed cells are present and thus mechanical lysis seems to be an appropriate method by which to further study frozen-thawed lysed cell removal.

Influence of the preimplantation embryo development (Ped) gene on embryonic platelet-activating factor (PAF) levels.

PURPOSE: A major gene responsible for the control of preimplantation cleavage rate is the Ped gene, the product of which is the Qa-2 protein. Fast, but not slow developing mouse embryos express the Qa-2 protein. Platelet-activating factor (PAF) is a novel and potent signaling phospholipid that has unique pleiotropic properties in addition to platelet activation. PAF plays a significant role in virtually every reproductive event, including ovulation, fertilization, implantation, and parturition. The role of the Ped gene in PAF production by preimplantation embryos is yet to be established. The presence of this gene provides embryos with a reproductive advantage over those that are Ped negative, and may also serve as a regulator of PAF synthesis. The study hypothesis is that the amount of PAF produced is dependent upon the presence or absence of the Ped gene. METHODS: B6.K1 (Ped negative) and B6.K2 (Ped positive) mouse embryo-conditioned culture media were assayed for PAF content by a PAF-specific radioimmunoassay. RESULTS: There was a significant (p < 0.001) difference in blastocyst development rates between the Ped+ B6.K2 (61.0%) and the Ped- B6.K1 (25.3%) embryo culture groups. There was a significant difference (p < 0.05) in PAF production between the Ped+ B6.K2 (4.70+/-0.46 pmol per embryo) embryo culture group and the Ped- B6.K1 (10.02+/-3.49 pmol per embryo) embryo group. The B6.K1 (Ped-) embryo group produced >2x more PAF than did the B6.K2 (Ped+) group. CONCLUSIONS: The Ped gene plays a role in PAF production and release in preimplantation stage embryos. The use of two mouse identical strains, except for the Ped gene, show that its presence is associated with an increase in developmental potential. Embryos where the Ped gene was absent produced significantly higher levels of PAF, which may aid in their survival.

Platelet-activating factor acetylhydrolase activity affects sperm motility and serves as a decapacitation factor.

OBJECTIVE: To determine the relationship between platelet-activating factor acetylhydrolase (PAFah) content in semen and sperm motility. DESIGN: The PAFah levels in semen were measured and correlated with sperm motility. SETTING: Clinical laboratory in a private assistant reproductive technology clinic. PATIENT(S): Three hundred and twelve men seeking diagnosis and treatment of infertility. INTERVENTION(S): Semen samples were collected from 312 healthy mature men seeking infertility treatment. Sperm motility and PAFah activity were measured in seminal plasma. Data was analyzed by Student's t test and regression analysis. MAIN OUTCOME MEASURE(S): PAFah activity and sperm motility. RESULT(S): Seminal PAFah content ranged from a low of 179 IU/L to a high of 2,457 IU/L. The overall mean PAFah content in semen was 780.59 IU/L. Linear regression analysis revealed a significant (R2 = 0.655) and negative relationship between PAFah content in semen and sperm motility. Semen specimens with high percent motility (> or = 50%) had significantly lower PAFah concentrations (442.03 +/- 14.37 IU/L) than those with the lower percent sperm motility (< 50%) (882.16 +/- 18.45 IU/L). CONCLUSION(S): The data confirm the presence of PAFah in human semen and that activity is significantly and negatively correlated with sperm motility. The PAFah is proven to be a candidate for sperm decapacitation factors, whereas PAF is qualified to be a candidate for sperm capacitation factors.

Comparison of laser-assisted hatching and acidified Tyrode's hatching by evaluation of blastocyst development rates in sibling embryos: a prospective randomized trial.

OBJECTIVE: To assess two zona drilling methods in terms of blastocyst development rates using sister embryos. DESIGN: Prospective, randomized study. Sister embryos of 14 patients were randomly assigned on day 3 to acidified Tyrode's zona drilling or to laser zona drilling. After biopsy, subsequent embryo culture until the blastocyst stage (day 5) was performed. SETTING: Private fertility center. PATIENT(S): Patients undergoing IVF-preimplantation genetic diagnosis. INTERVENTION(S): Embryo biopsy using either laser-assisted hatching or acidified Tyrode's hatching on sibling embryos and subsequent blastocyst development evaluation. MAIN OUTCOME MEASURE(S): Evaluation of blastocyst development in terms of degree of expansion and cell number in the inner cell mass and trophectoderm. RESULT(S): Blastocyst development rates (and blastocyst quality) were similarly high in both the acidified Tyrode's hatching group and the laser-assisted hatching group. CONCLUSION(S): Laser hatching does not impair embryonic development to the blastocyst stage, demonstrating that laser-assisted hatching is a suitable alternative to the use of acidified Tyrode's solution for zona drilling.

Effect of reduced oocyte aging on the outcome of rescue intracytoplasmic sperm injection.

OBJECTIVE: To evaluate the results of a novel protocol that allows to rescue IVF unfertilized oocytes by intracytoplasmic sperm injection (ICSI). DESIGN: Prospective clinical trial. SETTING: Private reproductive medical center. PATIENT(S): Thirty patients undergoing IVF. INTERVENTION(S): Controlled ovarian stimulation (COS), conventional IVF, rescue ICSI, embryo culture, and embryo transfer. MAIN OUTCOME MEASURE(S): Identification of unfertilized IVF oocytes 6 hours after insemination and fertilization, and developmental rates of those oocytes after rescue microinjection, as well implantation and pregnancy rates (PR). RESULT(S): All oocytes (392) from 30 patients were inseminated with standard IVF 3 hours after ovum pick-up. Polar body (PB) status was checked at decumulation and rechecked 3 hours later. Eighty-two oocytes were fertilized after IVF alone and 184 nonactivated oocytes (failed fertilization) were rescue microinjected and 166 of them fertilized (20 patients). Cleavage stage on day 2 was significantly more advanced and embryo grade was higher after standard IVF fertilization than after rescue ICSI. Eight of the 30 embryos transferred were implanted in the IVF-only patients (27%) and 8 of 68 embryos in the rescue ICSI patients (12%). CONCLUSION(S): Rescue ICSI of unfertilized IVF oocytes 6 hours after insemination (9 hours after egg retrieval) can provide normal fertilization, embryo development, and pregnancy; however, corresponding outcome parameters tend to be impaired in comparison to the standard IVF fertilization results.

High seminal platelet-activating factor acetylhydrolase activity in men with spinal cord injury.

Spinal cord injury (SCI) causes male infertility, with low sperm motility the major long-term cause. It has been suggested in previous studies that some seminal components may be responsible for the pathological asthenozoospermia. It is hypothesized that platelet-activating factor (PAF) acetylhydrolase (PAFah), which originates in the epididymis and other accessory sexual glands, may be a causative factor. This enzyme catalyzes PAF to acetate and biologically inactive lyso-PAF. PAF is well recognized to be an important phospholipid mediator that stimulates sperm motility and enhances sperm capacitation and fertilization. The present study was designed to analyze differences in PAFah activity in semen of men with SCI and age-matched healthy men. PAFah assay reagent kits were used to measure enzymatic activity by monitoring the production rates of 4-nitrophenol on a spectrophotometer during a given interval. The results showed that subjects with SCI had a higher concentration of PAFah than men in the control group (P < .001). A statistically significant negative correlation was found between enzymatic activity and sperm motility (r(2) = 0.8449; P < .001). Further studies will determine whether seminal vesicle dysfunction in men with SCI leads to abnormal PAFah activity, resulting in low sperm motility.

Impact of body mass index values on sperm quantity and quality.

Body mass index (BMI) has been demonstrated to affect female fertility; however, little information is available on the impact of BMI on male fertility or semen parameters. Therefore, the study objective was to determine the relationship between BMI and semen parameters, including sperm chromatin integrity. We analyzed data on semen samples from 520 men who were grouped based upon calculated BMI values (normal, 20-24 kg/m(2); overweight, 25-30 kg/m(2); obese, >30 kg/m(2)). The data collected included patient height and weight, semen volume, sperm concentration, percent sperm motility, percent sperm morphology (normal forms), and sperm chromatin integrity (DNA fragmentation index [DFI]). Data were analyzed by regression analysis and analysis of variance (ANOVA) with Tukey's test for multiple pairwise comparisons. The overall BMI mean (+/-SEM) was 27.5 (+/-0.49) kg/m(2). Linear regression revealed a significant (P < .05) and negative relationship between BMI and the total number of normal-motile sperm cells. ANOVA revealed a significant difference (P < .05) in the total number of normal-motile sperm cells among the different BMI groups. The number of normal-motile sperm cells per BMI group was as follows: normal, 18.6 x 10(6); overweight, 3.6 x 10(6); and obese, (0.7) x 10(6). All multiple pairwise comparisons were found to be significantly (P < .05) different. The overall DFI mean (+/-SEM) was 24.7 (+/-2.57). Linear regression revealed a significant (P < .05) and positive relation between BMI and DFI. Men presenting with a BMI greater than 25 kg/m(2) have fewer chromatin-intact normal-motile sperm cells per ejaculate. Therefore, to ensure maximum fertility potential, patients may be advised to reduce body weight.

Removal of lysed blastomeres from frozen-thawed embryos improves implantation and pregnancy rates in frozen embryo transfer cycles.

OBJECTIVE: To evaluate the effect of degenerated (lysed) blastomere removal on implantation and pregnancy rates in cleavage-stage cryo-embryo transfer (ET) cycles. DESIGN: Randomized clinical trial. SETTING: Private reproductive medical center. PATIENT(S): A total of 88 patients who received frozen-thawed ET, divided into two groups. INTERVENTION(S): Embryo freezing and thawing; opening of the zona pellucida and removal of cryodamaged blastomeres (in the study group), followed by same-day ET. MAIN OUTCOME MEASURE(S): Extent of survival of cleavage-stage embryos after the freeze-thaw procedure; embryo implantation and clinical pregnancies. RESULT(S): Oocyte number per patient, fertilization rate, embryo development rate (and quality), and freezing rates were similar in the two groups in the fresh cycle. In the control group, a total of 55 embryos (25%) of the 217 thawed remained fully intact, and 53 (26%) of the 207 in the study group remained intact. The average number of embryos transferred per group was similar (control, 3.4 +/- 0.9; study, 3.3 +/- 0.9). Implantation rates were 12% and 26% in the control and study groups, respectively. The clinical pregnancy rate was 23% in the control group and 64% in the study group when lysed cell removal was performed. CONCLUSION(S): The results show that pregnancy and implantation rates are higher in the study group; therefore, the removal of degenerated blastomeres may be beneficial to all patients who undergo cleavage-stage, frozen-thawed ET.

The significance of platelet-activating factor and fertility in the male primate: a review.

Since its discovery nearly 30 years ago platelet-activating factor (PAF) has emerged as one of the more important lipid mediators known. PAF (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphorylcholine) exists endogenously as a mixture of molecular species with structural variants of the alkyl moiety. PAF is a novel potent signaling phospholipid that has unique pleiotropic biological properties in addition to platelet activation. PAF also plays a significant role in reproduction and is present in the sperm of a number of primate species. PAF content in squirrel monkey sperm is significantly higher during the breeding season than the non-breeding season. PAF content in rhesus sperm has a significant relationship with sperm motility. PAF content in human sperm has a positive correlation with seminal parameters and pregnancy outcomes. The enzymes (lyso-PAF-acetyltransferase and PAF-acetylhydrolase) necessary for PAF activation and deactivation are present in primate sperm. PAF-acetylhydrolase may act as a "decapacitation factor". Removal of this enzyme during capacitation promotes PAF synthesis increasing primate motility and fertilization. PAF also plays a significant role in the fertilization process, enhancing the fertilization rates of oocytes. Enhanced embryo development has also been reported in oocytes fertilized with PAF-treated sperm. Exogenous PAF will also significantly improve primate artificial insemination pregnancy outcomes. PAF antagonists inhibit sperm motility, acrosome reaction, and fertilization thus suggesting the presence of receptors for PAF. The PAF-receptor is present on primate sperm, with altered transcript levels and distribution patterns on abnormal cells. Whereas, the exact mechanism of PAF in sperm function and reproduction is uncertain, its importance in normal primate fertility is substantial.

Exposure of preimplantation embryos to platelet-activating factor increases birth rate.

PROBLEM: Platelet-activating factor (PAF) plays a significant role in fertility. Preimplantation stage embryos produce PAF (ePAF) which is required for development. PAF's mechanism of action is receptor-mediated and its presence has been reported in the developing mouse and human embryo. Exposure of preimplantation stage mouse embryos results in higher implantation rates. However, the effect of such treatment on live-birth rates and birth weights has not been reported. Therefore, the objective the study was to determine the effect of exposing preimplantation mouse embryos to PAF on subsequent birth rate and weight. DESIGN: Two-cell stage preimplantation stage mouse embryos exposed to PAF (10(-7) M) for 15 min prior to intraoviductal transfer. METHODS: Preimplantation stage embryos were recovered from eCG/hCG primed BDF1 female mice. Embryos were exposed to synthetic PAF (10(-7) M) for 15 min. PAF-treated embryos were transferred to the oviducts of pseudopregnant female CD-1 female mice. Superovulated and cultured BDF1 embryos not treated with PAF served as in vitro controls and naturally ovulated embryos with no collection/culture served as in vivo controls. Embryos were permitted to develop to term (18-21 days). The number of pups born per litter and litter weights subsequently were recorded. RESULTS: A total of 160 BDF1 mouse embryos were collected, treated, and transferred (20 per CD-1 recipient) as described. There was a significant (P < 0.05) increase in the number of pups born to the PAF treatment group (56/80; 70%) as compared to the control group (44/80; 55%). There was also a significant difference (P < 0.05) in litter birth weights between the PAF (1.31 g/litter) and controls groups (1.25 g/litter). There was a significant difference (P < 0.05) in birth weights between the PAF treatment group and the in vivo group (1.51 g/litter). There was a significant difference in birth weights between the in vitro-control and in vivo groups (1.51 g/litter). There were no observational malformaties to pups born in any group. CONCLUSIONS: Brief exposure of preimplantation stage embryos to PAF will result in a significant increase of delivery rates (pups/litter) as well as birth weights. However, the increase of birth weight was significantly below that found naturally. Additional studies are warranted to elucidate the mechanism of PAF's action in the preimplantation stage embryo and subsequent uterine development.

Platelet-activating factor significantly enhances intrauterine insemination pregnancy rates in non-male factor infertility.

OBJECTIVE: To determine the efficacy of treating semen specimens with platelet-activating factor (PAF) before IUI. DESIGN: Prospective randomized double-blinded study of PAF treatment of sperm for patients with a history of infertility undergoing IUI. SETTING: Private infertility center. INTERVENTION(S): Patients had ovulation induction therapy with clomiphene citrate (CC) or gonadotropin, two IUIs per month with PAF treatment. MAIN OUTCOME MEASURE(S): Clinical pregnancy rates. RESULT(S): There was a significant difference in IUI pregnancy rates per cycle between control (10/56; 17.9%) and PAF (14/47; 29.8%) treatment groups in the normal male study arm. There was a significant difference in cumulative IUI pregnancy rates between control (10/35; 28.6%) and PAF (14/26; 53.9%) patient groups in the normal male study arm. There was no significant difference in IUI pregnancy rates per cycle between control (12/124; 9.7%) and PAF (14/119; 11.8%) treatment groups in the male factor study arm. There was no significant difference in cumulative IUI pregnancy rates between control (12/46; 26.1%) and PAF (14/38; 36.8%) patient groups in the male factor study arm. There was a significant difference in overall cumulative IUI pregnancy rates between control (21/81; 25.9%) and PAF (27/64; 42.2%) patient groups. CONCLUSION(S): The inclusion of PAF into the IUI sperm wash procedure significantly improves pregnancy rates. However, the significant improvement can only be shown to affect men presenting with normal semen parameters.