Oocyte Cleavage Research Articles

Levels of Steroid-Binding Proteins and Steroids in Human Preovulatory Follicle Fluid and Serum as Predictors of Success inin VitroFertilization-Embryo Transfer Treatment*

In an attempt to identify the embryos and cycles that have the best chances of resulting in establishment of pregnancies, after in vitro fertilization-embryo transfer (IVF-ET) treatment, the concentrations of sex hormone-binding globulin (SHBG) and cortisol-binding protein (CBP) were measured, using two new enzyme-linked immunosorbent assays in serum and follicle fluid (FF) from 30 women (125 FF) undergoing IVF-ET. The concentrations were compared to those of total estradiol and total progesterone, and correlated to oocyte cleavage and the establishment of pregnancies. Serum concentrations of CBP were significantly higher in women who became pregnant (1469 +/- 108) nM (+/- SEM] than in those who did not (CBP, 1200 +/- 58 nM; P less than or equal to 0.05). The concentrations of SHBG were not significant different in these two groups of women (72.4 +/- 9.3 and 60.8 +/- 4.2 nM, respectively; P greater than or equal to 0.10). By contrast, in FF significantly higher concentrations of both SHBG and CBP were found in women achieving pregnancy (SHBG, 56.1 +/- 2.8 nM; CBP, 1198 +/- 37 nM) than in those who did not (SHBG, 45.5 +/- 1.4 nM; P less than or equal to 0.001; CBP, 1079 +/- 29 nM; P less than or equal to 0.01). A positive correlation was found between serum and FF levels of both SHBG (r = 0.85; P less than or equal to 0.001) and CBP (r = 0.70; P less than or equal to 0.001). FF levels of estradiol and progesterone did not differ regardless of whether the oocyte cleaved. However, a significant reduction of estradiol was found in fluid from follicles in which the oocyte cleaved and resulted in pregnancy (3046 +/- 180 nM) than in fluid from follicles in which the oocyte cleaved but without establishment of pregnancy (4162 +/- 282 nM; P less than or equal to 0.001). There was no correlation between estradiol and SHBG and between progesterone and CBP. However, levels of FF progesterone above 15,000 nM combined with CBP concentrations above the mean concentration found in FF (1,127 nM) were related with oocyte cleavage in 87% of the cases. The overall cleavage rate is 56%. The higher levels of SHBG and CBP in serum compared to those in FF, and the positive relationship between serum and FF levels suggest that both proteins arise from the circulation. The similar levels in serum and FF indicate that neither SHBG nor CBP is responsible for maintaining the concentration gradient of estradiol and progesterone from follicle to plasma.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of heparin dosage and sperm capacitation time on in vitro fertilization and cleavage of bovine oocytes matured in vitro

The present study was conducted to clarify the effect of heparin dosage and sperm capacitation time on in vitro fertilization (Experiment 1) and cleavage (Experiment 2) rates of bovine oocytes matured in vitro. For in vitro fertilization, seven dosages of heparin (0, 5, 10, 25, 50, 100 and 200 μg/ml) and nine incubation periods (0, 5, 15, 30, 45, 60, 120, 180 and 240 min) in a capacitation medium were examined, using 6,634 oocytes. The mean proportions of fertilized oocytes in 25, 50 and 100 μg/ml of heparin were significantly (P<0.05) higher (53 to 59%) than in the other dosages (3 to 44%). Incubation with heparin for longer than 60 min lowered the frequencies of fertilization (20 to 36%) compared with the shorter incubation periods (38 to 49%). Higher proportions of fertilized oocytes were obtained by 5, 15, 30 or 45 min of incubation (42 to 49%) than by the other time periods (20 to 38%). Cleavage rates were found by using 2,098 oocytes in a factorial study (4 × 4 × 15: dosages −25, 50, 100 and 200 μg/ml; incubation periods −0, 15, 30 and 60 min; and replicates). The incubation periods and replicates resulted in highly significant differences (P<0.001) in development rates to eight-cell stage, but the four dosages of heparin showed no significant differences. The present results indicate that heparin dosage and sperm capacitation time are important factors influencing in vitro fertilization and cleavage rates. Optimal heparin dosages for the capacitation of bull spermatozoa ranged from 25 to 100 μg/ml; optimal incubation periods ranged from 5 to 60 min.

The Effect of the Follicular Fluid Adenosine 3′,5″-Monophosphate Degradation Rate on Successful Fertilization and Cleavage of Human Oocytes

Follicular fluid (FF) and oocytes were obtained from 19 women for in vitro fertilization. Ovulation was induced with clomiphene citrate and human menopausal gonadotropin. Thirty-seven FF samples containing mature oocyte-corona-cumulus complexes were used to measure steroids, gonadotropins, and cAMP. The FF specimens were divided into three groups: follicles yielding ova that were fertilized and cleaved (group A), follicles containing fertilized ova without further cleavage (group B), and follicles containing nonfertilized oocytes (group C). The FF levels of progesterone, 17 beta-estradiol, LH/hCG, and FSH did not differ significantly among the groups. Mean FF cAMP concentrations declined from 17.5 +/- 2.5 pmol/mL 15 min after follicle aspiration to 3.6 +/- 0.9 pmol/mL after 3 h. The initial FF cAMP concentration did not differ significantly among three groups. However, the cAMP degradation rate in group A (119 +/- 13 X 10(-4) pmol/min) was significantly greater than that in group B (62 +/- 10 X 10(-4) pmol/min) or group C (75 +/- 8 X 10(-4) pmol/min). In conclusion, an increased intrafollicular cAMP degradation rate was associated with successful fertilization and cleavage of human oocytes in vitro. These data suggest that the degradation rate of FF cAMP may be a marker of optimal follicular development and oocyte maturation.

In vitro fertilization and cleavage of bovine oocytes matured in medium supplemented with estrous cow serum

Bovine follicular oocytes were matured in TCM 199 supplemented with: (1) fetal calf serum (FCS, 20% v/v), Luteinizing Hormone (LH, 10 ug/ml), and Estradiol-17-β (E 2, 1 ug/ml) in Experiment 1; (2) 20% cow serum recovered at standing estrus (Experiment 2); or (3) 20% FCS (Experiment 3). Maturation, fertilization, and initial cleavage development were evaluated at 16 and 48 h after in vitro insemination. The proportions of oocytes fertilized after maturation in the presence of added hormones (78.5%, Experiment 1) or estrous serum (71.3%, Experiment 2) were significantly higher (p < 0.01) than after use of FCS alone (39.3%, Experiment 3). Cleavage of zygotes within 48 h post-insemination differed significantly (p < 0.01) between maturation treatments, 27.3%, 75.5% and 6.6% for Experiments 1, 2, and 3, respectively. Results demonstrate a beneficial influence of estrous cow serum, characterized by an elevated concentration of LH, on bovine oocyte maturation in vitro.

Effects of sera, hormones and granulosa cells added to culture medium for in-vitro maturation, fertilization, cleavage and development of bovine oocytes.

Sera (fetal calf serum: FCS; and oestrous cow serum: ECS), hormones (2.5 FSH micrograms/ml + 5 micrograms LH/ml + 1 microgram oestradiol/ml) and granulosa cells (5 x 10(6)/ml) were added to culture medium to determine the frequencies of in-vitro maturation, fertilization, cleavage (2- to 8-cell) and development into blastocysts of bovine follicular oocytes. The maturation rates after 24 h in culture were not significantly different among the three factors tested (56-72%). The fertilization rates were significantly affected by serum type and the addition of granulosa cells. FCS gave significantly higher rates of fertilization (57-71%) than did ECS (34-52%), but the proportions of polyspermic fertilization were significantly higher in the former (8-19%) than in the latter (2-3%). The addition of hormones did not affect fertilization, cleavage and development. Neither type of serum affected cleavage and development. The highest rates of blastocyst formation were obtained when granulosa cells alone were added (FCS, 17%; ECS, 16%). The cell numbers of the blastocysts obtained were 100-150, similar to those of blastocysts developed in vivo. Transfer of 6 blastocysts to 3 cows resulted in 1 pregnancy. The present results indicate that the co-culture with granulosa cells is the most important factor for in-vitro fertilization to development into blastocysts of bovine oocytes matured in vitro.

Lack of effect of high-dose antioestrogen on the maturation and in-vitro fertilization of human oocytes.

Thirty-four women requesting laparoscopic sterilization underwent a fixed schedule regimen for multiple follicular development which included norethisterone and clomiphene citrate. Follicle aspiration for oocyte recovery was attempted laparoscopically 34 h after administration of 5000 IU human chorionic gonadotrophin (HCG). Nineteen women were given 80 mg tamoxifen orally 4 h prior to HCG injection, while 15 acted as controls. There was no statistical difference in fertilization rates in vitro between tamoxifen-treated patients and controls (80 and 68% respectively). In addition, the morphological characteristics of the oocytes, the rates of cleavage, and the concentrations of oestradiol, progesterone and androstenedione in follicular fluid were similar in the two groups. Tamoxifen was detected in substantial amounts in follicular fluids of patients given tamoxifen. These results suggest that high-dose tamoxifen, in clinically used doses, does not adversely affect the final stages of maturation or the fertilization and early cleavage of human oocytes.

Fertilization and cleavage of mouse oocytes exposed to the conditions of human oocyte retrieval for in vitro fertilization*

AbstractOva from two strains of mice (a hybrid-inbred strain, B6D2F1, and a random-bred strain, CD1) were shocked by exposure to environmental conditions possibly encountered by human oocytes retrieved for in vitro fertilization (IVF). Shocked and control mouse ova were fertilized in vitro in either simple or complex media and zygote development to morulae and blastocyst stages compared with that of zygotes fertilized in vivo. Development of the hybrid-inbred zygotes following fertilization in the simple media of shocked and control ova was essentially the same as for ova fertilized in vivo (84 ± 6.6, 89 ± 1.6, 87 ± 6.0% to the 2-cell stage and 89 ± 5.2, 94 ± 2.3, 99 ± 1.0% of two cells to blastocysts, respectively); development in the complex media also was the same following fertilization of shocked and control ova (80 ± 8.7, 90 ± 2.7% to two cells and 33 ± 3.5, 35 ± 4.5% of two cells to blastocysts, respectively) but lower than that of in vivo zygotes (92 ± 4.6 to two cells, 58± 4.7 two cells to blastocysts). In contrast, the fertilization and development in the simple media of shocked and control random-bred ova was lower and more variable (90 ± 5.8, 67 ± 11.1% to two cells and 17 ± 8.3, 42 ± 13.6% two cells to blastocysts, respectively) than the development of in vivo zygotes (96 ± 1.5% to two cells, 51 ± 5.5 two cells to blastocysts). Thus, in these studies, fertilization and development of ovulated mouse ova were more dependent on the strain of mouse and the culture media conditions than on transient environmental shocks encountered before fertilization.

Epidermal growth factor (EGF) in human preovulatory follicles

Concentrations of epidermal growth factor (EGF), progesterone (P4) and oestradiol (E2) were determined in follicular fluid (FF) aspirated from 69 preovulatory follicles in 15 women undergoing ovarian hyperstimulation and ovulation induction as part of IVF-ET treatment for infertility. Sixty-nine oocytes were obtained from these FF aspirates and 35 (51%) fertilized and cleaved in vitro. Pregnancy was achieved in five (33%) of the women. Concentrations of EGF in FF ranged between 0.60 and 2.42 ng/ml, corresponding to approximately 50% of the level in serum at the time of follicle aspiration. A significant linear correlation between levels of EGF in FF and in serum was demonstrated. By contrast, no significant relationships were found between intrafollicular levels of EGF and those of P4 and E2, or between EGF in FF and oocyte cleavage rate in vitro and the achievement of pregnancy after ET. It is concluded that EGF in FF of the human preovulatory follicles reflects a passive diffusion into the follicle from the circulation. Moreover, that intrafollicular EGF is not involved in the local regulation of human preovulatory follicle and oocyte maturation. These results, however, do not preclude an auto- and/or paracrine role of intrafollicular EGF in smaller human follicles.

Wheat germ agglutinin treatment of follicle cell-free mouse oocytes inhibits fertilization.

Controversy exists whether treatment of follicle cell-free oocytes with wheat germ agglutinin (WGA) prevents fertilization. Lack of inhibition in one case has led to the suggestion that acrosin may not be a zona lysin. To re-examine the effect of the WGA, the zona pellucida of follicle cell-free mouse oocytes was made more resistant to proteinase digestion by treatment with 10 or 50 micrograms/ml WGA. Such WGA-treated oocytes showed decreased fertilizability when washed to remove excess WGA and incubated with capacitated spermatozoa. Oocyte cleavage was used as an end point, because a large number of spermatozoa adhered to the eggs after WGA treatment, making observation of sperm penetration and pronucleus formation unreliable. Resistance to proteinase digestion increased, and the fertilizability decreased with the higher amount of WGA. The action of WGA was most likely not mediated by a direct effect on sperm motility, sperm acrosin activity, sperm binding to the zona pellucida, or oocyte cleavage. WGA did not affect the acrosome reaction of guinea pig spermatozoa. These data show that WGA treatment of follicle cell-free mouse oocytes results in decreased fertilizability, possibly by rendering the zona pellucida more resistant to sperm proteinase digestion.

Correlation of pregnancy after gamete intrafallopian transfer with fertilization and cleavage of sibling oocytes

In this study, GIFT resulted in a 44% pregnancy rate. When excess oocytes fertilized and developed to the blastocyst stage, the pregnancy rate was 78%. The continuing pregnancy rate was increased from 31% to 55% when sibling blastocysts developed.

The cell cycle can occur in starfish oocytes and embryos without the production of transferable MPF (maturation-promoting factor)

All cells undergoing the transition from interphase to metaphase have been postulated to contain a “maturation-promoting factor” (MPF) capable of causing meiotic maturation when injected into immature oocytes. We have shown in an accompanying paper ( A. Picard, M. C. Harricane, J. C. Labbe, and M. Doreé, 1988, Dev. Biol. 128, 121–128) that the basic oscillator driving the cell cycle still operates in maturing starfish oocytes and fertilized eggs in the absence of germinal vesicle (GV) material. Under such conditions of enucleation, we now show, however, that MPF activity cannot be detected after hormonal stimulation of prophase-arrested oocytes in Astropecten or after the normal time of second meiotic cleavage in Marthasterias. In contrast, cell cycles occur with the production of transferable MPF activity in embryos from which both pronuclei have been removed after fertilization. Reinjection of the entire contents of a GV after the normal time of second meiotic cleavage restores the ability of cytoplasm to induce meiotic maturation in immature recipient oocytes after transfer. Transduction of the hormonal stimulus at the level of the plasma membrane, stimulation of the phosphorylation of cytoplasmic proteins, and activation of a cycling Ca 2+- and cyclic nucleotide-independent histone kinase still occur in the absence of GV material. Since previous studies have demonstrated that the presence of GV material in the recipient oocytes is absolutely required in starfish for the amplification of microinjected MPF ( Kishimoto et al., 1981 ; Picard and Doree, 1984), we propose that some unidentified component of the GV is required, at least after the normal time of second meiotic cleavage in donor oocytes and at any time in recipient oocytes, for the successful transfer of MPF activity in starfish.

RNA 3′ cleavage and polyadenylation in oocytes and unfertilized eggs of Xenopus laevis

Xenopus laevis histone H4 and H1 genes were transcribed in vitro to generate artificial precursor mRNAs (pre-mRNAs). These pre-mRNAs were microinjected into oocytes, matured oocytes, and unfertilised eggs of Xenopus laevis and their 3′ cleavage and polyadenylation were investigated. In the oocyte nucleus both H4 and H1 pre-mRNAs were 3′ cleaved but were not detectably polyadenylated. In the oocyte cytoplasm there was neither 3′ cleavage nor polyadenylation of these histone pre-mRNAs. When injected into either matured oocytes or unfertilised eggs, the pre-mRNAs underwent 3′ cleavage but this was inefficient when compared to the oocyte nucleus. In addition approximately 50% of the remaining uncleaved pre-mRNA was subject to a polyadenylation activity which added A tails of approximately 70 A residues. In contrast, artificial mouse β-globin pre-mRNAs were not detectably 3′ cleaved or polyadenylated in either microinjected oocytes or unfertilised eggs.

Lipid and lipoprotein contents of human follicular fluid.

The concentrations of total cholesterol, phospholipids, triacylglycerols and lipoproteins were measured in 87 follicular fluids obtained from 35 women undergoing in vitro fertilization and embryo transfer. The results were correlated with the levels of progesterone in follicular fluid. Two different types of ovarian stimulation were used. High density lipoproteins were the dominant lipoproteins found in the preovulatory follicular fluid. Low density lipoproteins were absent or appeared in trace amounts. Significantly higher triacylglycerol and high density lipoprotein levels were found when stimulation with human menopausal gonadotropins and chorionic gonadotropin was applied, as compared to the clomiphene citrate-menopausal gonadotropin-chorionic gonadotropin menstrual cycle. In both groups, extracorporal fertilization resulted in cleavage of oocytes and embryo transfer. No significant correlation between any follicular fluid lipid and progesterone concentration was found. The lipids estimated in the follicular fluid appeared to have no influence on the oocyte fertilizability. The presence of triacylglycerols and high density lipoproteins in the follicular fluid may indicate follicular wall permeability under the treatment with menopausal gonadotropins.

Ram-specific effects on in-vitro fertilization and cleavage of sheep oocytes matured in vitro.

The present experiments were designed to identify possible male-specific effects on early embryonic development in vitro: Sheep oocytes were matured in vitro for 24-26 h and then fertilized in vitro using equal numbers of viable spermatozoa from 1 of 6 Clun Forest rams. At 15-18 h after insemination, oocytes were either fixed and examined for fertilization and polyspermy or further cultured in modified M 199 medium for 3 days in an oviduct epithelial co-culture system. There were significant differences in 5 separate trials between the rams with respect to the rate of fertilization, degree of polyspermy and cleavage rate after monospermic fertilization. The mean rate of fertilization varied from 89% in Ram B to 43% in Ram C while the percentage of polyspermic eggs varied from 5 to 34%. Both the absolute number of embryos cleaving to the 16-cell stage and the calculated percentage of monospermic eggs reaching the 16-cell stage differed markedly between groups of eggs fertilized by different rams. The results indicate that the development of sheep eggs in vitro is differentially affected by the ram from which the spermatozoa are collected.

Effect of general anesthesia on fertilization and cleavage of human oocytes in vitro

To determine whether anesthesia affects in vitro fertilization (IVF), the authors examined 3 1/2 years' experience with IVF. Anesthesia length significantly predicted fertilization and cleavage at stage 0 of stepwise multiple logistic regression analysis, but not at the final step. Oocyte grade, retrieval order, and a quadratic term for grade remained significant for fertilization; cleavage, order, the interaction of order and grade, and the quadratic term for grade remained significant. Order correlated with anesthesia (r = 0.675, P less than 0.001). Also inherent in order are CO2 pneumoperitoneum, increased prolactin, decreased gonadotropins, ovarian trauma, and time. First oocytes of equivalent grade from contralateral ovaries were compared. Fertilization rates were equivalent, but significantly fewer mature oocytes from the second ovary cleaved. Anesthetic agents and CO2 appear to adversely affect fertilization and cleavage in vitro.

Fertilization and embryo development of oocytes obtained transvaginally under ultrasound guidance.

The widespread use of ultrasound for follicle development assessment and ovum pickup has raised concerns about the theoretical possibility of adverse effects on oocyte function. Studies in other species do not demonstrate any adverse effects when using sonic energies as those presently used in diagnostic ultrasound. However, no data are available concerning the effects of diagnostic ultrasound on human preovulatory oocytes. This study was conducted to determine whether there were any adverse effects on the reproductive function of oocytes using transvaginal ultrasonography. No effect on fertilization and subsequent cleavage of oocytes was demonstrated. This method can, therefore, be recommended for follicle assessment and as a better alternative to laparoscopy for oocyte retrieval.

Relationship of human oocyte maturity, fertilization, and cleavage to follicular fluid prolactin and steroids.

Follicular fluid (FF) levels of prolactin (PRL), estradiol (E2), progesterone (P), and androstenedione (delta 4A) were related to diversities in oocyte maturation, fertilization, and cleavage among oocytes obtained for in vitro fertilization. Follicles with mature stage I oocytes contained similar concentrations of PRL and E2, more P, and less delta 4A compared to follicles containing immature oocytes. Follicles with mature stage II oocytes had higher amounts of P and E2 but similar levels of PRL and delta 4A compared to follicles with mature stage I oocytes. Prolactin was lower and E2 higher in FF associated with fertilizable oocytes than in those with unfertilized gametes. Follicles which yielded gametes that ultimately divided to more than four cells had the highest E2 levels, while the lowest E2 concentrations were associated with fertilized, uncleaved eggs. P, delta 4A, and PRL levels in FF were not related to oocyte cleavage. We conclude that human menopausal gonadotropin (hMG) treatment does not necessarily lead to a synchrony or uniformity of preovulatory follicles, maturation of oocytes is associated with rising levels of progesterone and decreasing levels of delta 4A in FF, an increased cleavage rate in vitro is associated with higher FF levels of E2, and high levels of PRL in FF are associated with unfertilized oocytes. These findings emphasize that differences in the hormonal milieu surrounding oocytes may have profound effects on the success of in vitro fertilization.

Follicle culture enhances fertilizability and cleavage of bovine oocytes matured in vitro.

The fertilization and cleavage of bovine oocytes matured by intra- or extra-follicular methods were investigated. Oocytes were fertilized in vitro or in the rabbit oviduct and cleavage was assessed after in vitro culture of in vitro fertilized oocytes and after in vivo culture (rabbit oviducts) of xenogenously fertilized oocytes. The effect of fertilization with fresh-diluted or frozen-thawed semen were also examined. The intra-follicular method did not increase the nuclear maturation rate as compared with the extra-follicular method (57.9 and 52.7%, respectively). However, the proportions of in vitro fertilized eggs (54.8%) and of cleaved eggs (two to eight cells; 34.6%) in the rabbit oviduct for 48 h after xenogenous fertilization were higher (P less than .025) in the intra-follicular oocytes than those of the extra-follicular oocytes (37.1 and 21.3%, respectively). It was also found that the use of fresh-diluted semen resulted in more cleaved eggs from the rabbit oviduct than the use of frozen-thawed semen (43.4 and 23.3% in the intra-follicular oocytes, P less than .025; 31.0 and 7.8% in the extra-follicular oocytes, P less than .05), while the appearance of cleaved eggs following in vitro fertilization was extremely low (0 to 6.6%). The present results demonstrated that the intra-follicular culture method of bovine oocytes provided a physiological environment for cytoplasmic maturation leading to higher fertilizability and development than the conventional in vitro culture of extra-follicular oocytes.

Androgen and 19-norsteroid profiles in human preovulatory follicles from stimulated cycles: An isotope dilution-mass spectrometric study

Follicular fluid was aspirated from preovulatory follicles of women under ovarian stimulation for in vitro fertilization and analyzed by a highly specific technique based on gas chromatography-mass spectrometry associated with stable isotope dilution. 19-Nortestosterone and 19-noradrostenedione were identified and quantified for the first time in human follicular fluid. There was a strong positive correlation between 19-nortestosterone and estradiol-17β and between 19-norandrostenedione and estrone concentrations, thus indicating a common cellular origin. The accumulation of 19-norsteroids in follicular fluid confirms that they are weakly active intermediates in the multistep enzymatic conversion of androgen to estrogen. Testosterone concentrations were significantly lower than those obtained by radioimmunoassay; cross-reaction with substantially higher levels of 19-nortestosterone seems to be at the origin of this discrepancy. Androstenedione concentrations were similar to those reported in the literature and it was therefore confirmed that an estradiol/androstenedione concentration ratio above 20 is favourable for oocyte cleavage. Other and some newly estimated androgens are: testosterone sulfate, 5-androstene-3β,17β-diol 3-sulfate and disulfate, dihydrotestosterone sulfate, epitestosterone, 19-hydroxy-androstenedione, 5α-androstane-3α,17β-diol, 5α-androstane-3β,17β-diol, 5α-androstane-3,17-dione and androsterone. Dehydroepiandrosterone sulfate was by far the most abundant androgen in this type of follicles.

Incubator performance in the clinical in vitro fertilization program: importance of temperature conditions for the fertilization and cleavage of human oocytes

Incubator performance in the clinical in vitro fertilization program: importance of temperature conditions for the fertilization and cleavage of human oocytes