An in-vitro study of ginsenoside Rb1-induced teratogenicity using a whole rat embryo culture model

BACKGROUND: Ginseng is a commonly used herbal medicine worldwide. However, there is limited information regarding its effects on the developing embryo. METHODS: The effect of ginsenoside on the developing embryo during the critical period of organogenesis was investigated using a whole rat embryo culture model. Embryos were exposed to various concentrations of ginsenoside Rb(1) and scored for growth and differentiation at the end of the culture period. RESULTS: Median total morphological scores in embryos exposed to 30 micro g/ml of ginsenoside Rb(1) was significantly lower (P < 0.05) than that in control embryos (35 versus 45). Morphological scores for flexion, forelimb and hindlimb were also significantly reduced. The median total morphological scores further decreased to 28 when the concentration of ginsenoside Rb(1) was increased to 50 micro g/ml. At this concentration, the embryonic crown-rump length and somite number were also significantly reduced compared with control embryos (2.8 versus 3.0 mm and 16.0 versus 21.0, respectively). CONCLUSIONS: Our study has demonstrated that ginsenoside exerts direct teratogenic effects on rat embryos. Until more is known about the effects of ginsenoside in women of reproductive age, we suggest its use should be treated with caution.     

A study on placental transfer of diclofenac in first trimester of human pregnancy

Diclofenac is a commonly used non-steroidal anti-inflammatory drug in women of reproductive age. It has teratogenic effects in animals. The aim of this study was to investigate the placental transfer of diclofenac in the first trimester of human pregnancy. Thirty patients undergoing surgical termination of pregnancy between 8 and 12 weeks gestation were given two doses of diclofenac before the procedure. Corresponding samples of maternal serum, amniotic fluid, coelomic fluid and fetal tissue were analysed by high-performance liquid chromatography. Diclofenac was detectable in all fetal tissue samples, with a concentration similar to that found in maternal venous samples. However, diclofenac was detectable in only 56.7 and 23.3% of the coelomic and amniotic fluid samples respectively, and the highest concentration attained was 80 and 5% of the maternal concentration respectively. In summary, we confirmed that diclofenac crosses the human placenta readily during the first trimester. Further studies are required to investigate the potential teratogenic effect of diclofenac in human embryos.     

An in-vivo study on placental transfer of naproxen in early human pregnancy

BACKGROUND: Naproxen is one of the most common non-steroidal anti-inflammatory drugs used by women of reproductive age. Naproxen is known to be teratogenic in animals. The aim of this study was to investigate the placental transfer of naproxen in the first trimester of human pregnancy, and to determine the amount of the drug in different embryonic compartments. METHODS: Twenty-eight patients who requested surgical termination of pregnancy in the first trimester were given two oral 500 mg doses of naproxen before the surgical procedure. Four biological samples, maternal venous blood, coelomic fluid, amniotic fluid and fetal tissue, were collected from each patient for drug analyses by high performance liquid chromatography. RESULTS: Naproxen was detected in all samples. The mean (+/- SD) concentrations were 69.5 +/- 12.2 microg/ml, 6.4 +/- 2.4 microg/g, 1.85 +/- 1.03 microg/ml and 0.14 +/- 0.11 microg/ml in maternal serum, fetal tissue, coelomic fluid and amniotic fluid respectively. The mean amniotic fluid/maternal drug ratio and fetal/maternal drug ratio were 0.002 (range 0.0005-0.0064) and 0.092 (range 0.022-0.155) respectively. There was a positive correlation between the fetal drug concentration (r = 0.59, P = 0.001), amniotic fluid drug concentration (r = 0.47, P = 0.013), amniotic fluid/maternal ratio (r = 0.536, P = 0.003) and fetal/maternal ratio (r = 0.72, P < 0.001) with advancing gestational age. CONCLUSIONS: Although naproxen can cross the placenta readily in the first trimester of human pregnancy, only a small amount was present in fetal tissues. Since there is no information on whether this small amount of naproxen would be teratogenic or not, women of reproductive age who are taking naproxen regularly should be warned of the possible fetal side-effects.     

Culture of whole rodent embryos in teratogen screening

Rodent embryos can be grown in vitro during a period of rapid organogenesis. During a 24-48 hour culture period, growth and development closely approximate the values of embryos in utero at the same gestational stage. Embryos can be exposed to carefully controlled concentrations of test substances in a system which is free of maternal variables such as nutritional status or stress effects. Developmental abnormalities are limited to those systems developing during the culture period, but those available may show heightened sensitivity relative to the embryo in utero. Recently several systems have been developed which permit incorporation of either metabolites or metabolic enzymes in embryo culture. These materials can be obtained from species other than that of the test embryos. Because studies have shown the importance of maternal metabolism in the activation and inactivation of teratogens, it is hoped that these systems will enable us to understand the biochemical basis of species variability in teratogenic sensitivity and construct a teratogen screen which can reliably identify compounds which pose teratogenic hazards to humans.     

A study of teratogenicity of hydrosalpinx fluid using a whole rat embryo culture model

BACKGROUND: Hydrosalpinx fluid may be toxic to sperm and early embryo growth. Information concerning the effect of hydrosalpinx fluid on embryo development during organogenesis is lacking. METHODS: Rat embryos at gestational day 9.5 were cultured for 48 h with 80% rat serum and 20% of either hydrosalpinx fluid (study group) or lactated Ringer's solution (control group). Embryos were scored for growth and development at the end of the culture period. RESULTS: Hydrosalpinx fluid, collected from 10 patients, was tested for embryotoxicity individually. Median total morphological scores were significantly lower in embryos exposed to hydrosalpinx fluid from three of the 10 patients (43.0 versus 47.0, P = 0.01; 36.0 versus 45.0, P < 0.001; 36.0 versus 46.5, P = 0.003). This was accompanied by a significant reduction in median yolk sac diameter (4.0 versus 5.2 mm, P < 0.001 and 4.0 versus 5.0 mm, P < 0.001) and somite number (17.5 versus 22.5, P < 0.001 and 17.0 versus 21.5, P = 0.008) in the latter two patients. CONCLUSIONS: Hydrosalpinx fluid in some patients may contain toxin(s) that is potentially teratogenic.     

Teratogenic IgG From Sera of Women With Spontaneous Abortions Seem to Induce Anomalies and Yolk Sac Damage in Rat Embryos. A Possible Method to Detect Abortions of Immunologic Origin

PROBLEM: Spontaneous abortions due to immunological rejection of the embryo may be avoided by immunotherapy with paternal allogeneic leukocytes but there is no appropriate method to detect and differentiate this group of aborters from other groups. METHODS: In previous studies we have demonstrated that in about two-thirds of sera from women with spontaneous abortions the IgG antibodies are responsible (alone or in combination with other factors) for the embryotoxic effects of these sera on cultured rat embryos. We presently cultured 10.5-day-old rat embryos on highly teratogenic serum (“high risk” serum that induced anomalies in more than 50% of the embryos) from women with spontaneous abortions, where the IgG fraction was exchanged with IgG from control sera and vice-versa. We studied by Transmission Electron Microscopy (TEM) the extent of yolk sac damage in comparison to the rate of embryonic anomalies. RESULTS: In cases where IgG antibodies were teratogenic, embryos cultured in control sera with IgG from “high risk” sera exhibited ultrastructural yolk sac damage as well as embryonic anomalies, and the yolk sacs cultured in “high risk” sera with control IgG were normal. In cases in which the IgG exchange did not change the rate of anomalies, as IgG was not teratogenic, yolk sacs from embryos cultured in “high risk” sera remained damaged, while yolk sacs from embryos cultured in control sera after IgG exchange stayed normal. Although no significant difference in total IgG levels was found between the groups, a higher IgG1 level in sera from women with teratogenic IgG was observed in comparison to control women's sera. The obstetrical history of the women with two or more abortions who took part in our study showed that there were more cases of unknown etiology of the abortion in the women from the “high risk” group. CONCLUSIONS: The serum and the IgG fraction from women with habitual abortions can be tested in whole embryo culture to evaluate the embryonic and yolk sac damage. On this basis it may be possible to detect the women in whom the habitual abortions result from immunological rejection.     

Co-culture of rat embryos and hepatocytes: in vitro detection of a proteratogen

The technique of whole embryo culture developed by New [Environ Health Perspect 18:105-110, 1976] provides a sensitive assay to evaluate the effects of a test chemical on embryo development independent of maternal influences. To detect proteratogens, this assay must be coupled with an exogenous metabolic activation system. We have developed methods for the co-cultivation of rat embryos with primary hepatocytes, which offers several advantages over subcellular fractions when providing metabolic activation for in vitro assays. In the present study, rat embryos removed from the dam on day 10 of pregnancy were co-cultivated in vitro with primary cultures of rat, rabbit, or hamster hepatocytes. Embryos co-cultivated with hepatocytes developed normally, as did embryos exposed to a test chemical, cyclophosphamide (CP) in the absence of hepatocytes. When embryos were co-cultivated with hepatocytes and exposed to CP, a dose-related embryotoxicity was observed, indicating metabolic activation of the proteratogen. Using hepatocytes isolated from rats pretreated in vivo with phenobarbital, we observed an increase in CP-induced malformations and embryotoxicity compared to those of embryos exposed to CP in the presence of uninduced hepatocytes. The teratogenic bioactivation of CP was inhibited in vitro by the addition of metyrapone. When similar numbers of hepatocytes were used for metabolic activation of CP the induced embryotoxicity was greater in the presence of rabbit and hamster hepatocytes than with rat hepatocytes. Development of procedures for the culture of rat embryos with hepatocytes from other species suggests the utility of this in vitro system for the investigation of species differences in sensitivity to chemical teratogens.     

The Effects of Sera From Women With Spontaneous Abortions on the In Vitro Development of Early Somite Stage Rat Embryos

PROBLEM: Spontaneous abortions occur in 40 to 50% of pregnancies, but the causes for some abortions, especially those that are recurrent (spontaneous), are still unknown. METHOD: Following previous studies that demonstrated embryotoxic effects of sera from women with spontaneous abortions in preimplantation mouse embryos, we cultured 10.5-day-old rat embryos in sera from women after spontaneous abortions to look for specific teratogenic effects. RESULTS: About 50% of the embryos cultured in sera from women after spontaneous abortions were malformed, as compared to 19.1 and 27.1% malformations in embryos cultured in sera from women after a normal delivery and during a normal second trimester of pregnancy, respectively. We divided the sera from women who had spontaneous abortions into high-risk, and low-risk sera. In the high-risk sera from one abortion, we found 74.2% malformed embryos and in the high-risk group from two or more abortions this rate was 81.0%. This is compared to a rate of 17.1 and 10.3% in the low-risk sera, respectively. We have also found lower DNA and protein synthesis in the embryos cultured in high-risk sera compared to those cultured in low-risk and control sera. Transmission electron microscopy examination of yolk sacs cultured in high risk sera showed ultrastructural damage as represented by a lower number of microvilli and a higher number of inclusions in the entodermal cells when compared to controls. Amino acid chromatography of the serum and the concentrations of folic acid and zinc were similar in control and high-risk sera. CONCLUSION: It seems that the majority of sera from women with unexplained spontaneous abortions are teratogenic to rat embryos in culture. In about two-thirds of these sera the teratogenic factor(s) seem to be present in the IgG fraction.     

Teratogenesis after acute alcohol exposure in cultured rat embryos

In order to investigate whether alcohol has teratogenic properties, rat embryos were cultured in vitro during their organogenetic period and exposed to ethanol at 200-800 mg% culture medium for the 48 hours of culture (0-24 somite stage) or to 600-800 mg% for 24 hours, or 6-hour periods. Exposure to alcohol throughout the entire 48-hour culture period or the first 24-hour period (0-12 somites) produced marked growth retardation, particularly of the head region in a dose-dependent manner, but did not prevent neural tube closure. Exposure to high levels of ethanol during specific 6-hour periods of early organogenesis (three to nine somites) prevented closure of the neural tube in 30% of cultured rat embryos, indicating a direct teratogenic action of ethanol. These results implied an effect of ethanol on embryonic development, independent of maternal metabolism. The 6-hour exposure experiments demonstrated that high doses of ethanol during specific periods of organogenesis can be teratogenic.     

Granulocyte-macrophage colony-stimulating factor promotes human blastocyst development in vitro

The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is synthesized in the female reproductive tract and has been implicated in the growth and development of the preimplantation embryo in rodent and livestock species. To examine the effect of GM-CSF on human embryo development in vitro, surplus frozen 2-4-cell embryos were cultured in media supplemented with 2 ng/ml recombinant human GM-CSF. The addition of cytokine increased the proportion of embryos that developed to the blastocyst stage from 30 to 76%. The developmental competence of these blastocysts, as assessed by hatching and attachment to extracellular matrix-coated culture dishes, was also improved by GM-CSF. The period in culture required for 50% of the total number of blastocysts to form was reduced by 14 h, and blastocysts grown in GM-CSF were found to contain approximately 35% more cells, due primarily to an increase in the size of the inner cell mass. The beneficial effect of GM-CSF was exerted in each of two sequential media systems (IVF-50/S2 and G1. 2/G2.2) and was independent of the formulation of recombinant cytokine that was used. These data indicate that GM-CSF may have a physiological role in promoting the development of the human embryo as it traverses the reproductive tract in vivo, and suggest that addition of this cytokine to embryo culture media may improve the yield of implantation-competent blastocysts in human in-vitro fertilization programmes.     

Live-birth rates and multiple-birth risk of assisted reproductive technology pregnancies conceived using thawed embryos,USA 1999-2000

BACKGROUND: Increasing use of assisted reproductive technology treatments has been associated with the current rise in multiple births in the USA. Embryo cryopreservation and subsequent thawed embryo transfer may favourably impact the multiple-birth risk by relieving some pressure that patients and providers may feel to transfer several embryos in a single cycle. The study objective was to examine both live-birth rates and multiple-birth risk in thawed cycles. METHODS: The authors used a population-based sample of 21 555 assisted reproductive technology procedures performed in US clinics in 1999 and 2000 that used thawed embryos derived from the patient's oocytes. RESULTS: Both patient age and the number of embryos transferred were independent predictors of live birth. Even among women aged 20-29 years, the transfer of three embryos resulted in an increase in the live-birth rate compared with cycles in which one or two embryos were transferred. This increase in success was accompanied by an increased multiple-birth risk. In all age groups up to 40 years, the transfer of just two embryos resulted in a multiple-birth risk of 16-17%. The multiple-birth risk increased with the number of embryos transferred. CONCLUSIONS: Patient age and the number of embryos transferred significantly affect live-birth and multiple-birth rates among women who use thawed embryos.     

Potentiation of the maternal immune system may modify the apoptotic process in embryos exposed to developmental toxicants

PROBLEM: We have previously shown that teratogen-induced embryonic maldevelopment may result from excessive apoptosis in affected organs, but the mechanisms underlying this process are not well understood. Here we investigate the ability of maternal immunopotentiation to affect the apoptotic process and its regulatory genes p53 and bcl-2 in embryos exposed to a teratogenic insult. METHOD OF STUDY: Potentiation of the immune system in pregnant females was performed with xenogeneic rat splenocytes or with granulocyte macrophage-colony stimulating factor (GM-CSF). The animals were exposed to cyclophosphamide (CP) and the reproductive performance in the various experimental groups was recorded. The level of apoptosis was assessed in the embryonic head and liver by TdT-mediated dUTP-biotin nick end labeling and fluorescence-activated cell sorter (FACS) analysis, while p53 and bcl-2 expression was evaluated by FACS and immunohistochemistry. RESULTS: In CP-treated females, a decrease in embryonic weight and an increase in the resorption rate and the percentage of embryos exhibiting head malformations were noted. These effects of CP were accompanied by the appearance of apoptotic cells in the head but not in the liver and an increased expression of p53 in embryonic organs, while bcl-2 expression was found to be decreased in the head and increased in the liver. Immunopotentiation with rat splenocytes or GM-CSF was shown to partially normalize the teratogenic effect of CP. It was also found to partially decrease the CP-induced apoptotic process and exhibited a tendency to normalize the expression of p53 and bcl-2 in the embryonic head and liver. CONCLUSION: Our results suggest a possible role for maternal immunopotentiation in protecting the embryo from teratogenic insults, possibly through regulation of the CP-induced apoptotic process and the expression of p53 and bcl-2.     

Embryotoxicity of the pesticide mirex in vitro

Mirex is a pesticide that is environmentally stable, accumulates in body tissues, and is embryo- and feto-toxic at high concentrations in vivo. This study is the first to evaluate the effects of mirex on organogenesis-stage embryos in vitro. Mouse embryos were exposed on gestation day 8.5 for 24 h in whole-embryo culture to mirex at 100, 200, or 400 microg/ml dissolved in xylene and compared with xylene-treated controls (1, 2, or 4 microl/ml, respectively) and untreated controls. Embryos were evaluated for malformations, somite number, total protein content, and visceral yolk sac circulation. Potential embryotoxic mechanisms were evaluated by using PCNA stain for cell proliferation and the TUNEL assay for apoptotic cell death. Mirex-exposed embryos demonstrated increased malformation rates and decreased total embryonic protein contents at > or =200 microg/ml mirex, and decreased somite numbers and VYS circulation at > or =100 microg/ml mirex, compared with xylene-treated controls. There was no difference in PCNA levels or TUNEL staining in mirex-treated embryos compared with xylene-treated controls or untreated controls. Thus, mirex is embryotoxic in vitro to early organogenesis stage mouse embryos at concentrations > or =100 microg/ml, but the effects do not appear to be mediated by changes in cell proliferation or apoptotic cell death.     

Assisted hatching improves implantation rates on cryopreserved-thawed embryos. A randomized prospective study

BACKGROUND: Focus on the hatching process has so far been in the field of fresh embryos. Cryopreserved-thawed embryos have a lower rate of pregnancy than fresh embryos, which might be due to hardening of the zona pellucida. METHODS: During a 2 year period, a prospective randomized study enrolling 253 cryopreserved-thawed cycles was performed on day 2 embryos. Pseudorandomization to assisted hatching or a control group was done on the basis of even and odd dates for thawing. One hour before embryo transfer, hatching was carried out using acidic Tyrode's solution. RESULTS: Among 136 embryos exposed to assisted hatching, 11.4% (30) were implanted compared with only 5.8% (13) of 117 embryos not exposed to assisted hatching (P<0.05, chi(2) test). No difference in the rate of clinical pregnancy and positive serum HCG was observed between the two groups. Very few women >38 years old were included in the study, and no significant difference according to age could be found between the groups. CONCLUSIONS: These results show that assisted hatching using acidic Tyrode's solution increases the implantation rate of cryopreserved-thawed embryos (P<0.05).     

Impact of hyperglycemia on early embryo development and embryopathy: in vitro experiments using a mouse model

BACKGROUND: The aim of this study is to model the processes of early embryopathy seen in human pregnancy complicated by maternal hyperglycemia secondary to maternal diabetes using a mouse embryo culture system. METHODS: Female mice were superovulated and mated in pairs. Two-cell embryos were harvested from the oviducts and cultured in vitro in KSOM medium (synthetic oviductal medium enriched with potassium) supplemented with 0.2, 5.56, 15.56 or 25.56 mM d-glucose. Cell proliferation, differentiation and apoptosis were assessed. Experiments were performed in constant, embryos exposed to a particular concentration of glucose (0.2, 5.56, 15.56 or 25.56 mM) from harvest to either Day 5 post fertilization (pf) or Day 8 pf, and fluctuating, embryos exposed to alternate high 25.56 mM and normal 5.56 mM concentrations of glucose between harvest and Day 5 pf, glycemic culture. RESULTS: Expected levels of blastocyst formation and hatching were seen at 0.2 and 5.56 mM concentrations of glucose but both were impaired at higher concentrations (chi(2), P < 0.005; P < 0.001). Total cell numbers (P < 0.002) and cell allocation to the inner cell mass (P < 0.01) were reduced, but with no evidence of enhanced apoptosis in the hyperglycemic cultures. Variation in hyperglycemic exposure of the embryos on Days 2, 3 and 4 showed no adverse effects of hyperglycemia up to 24 h, but 48 and 72 h exposures were equally embryopathic (P < 0.01). CONCLUSIONS: Hyperglycemic exposure for >24 h is toxic to early embryo development. These findings may explain the lower than expected implantation rates and higher than expected rates of congenital abnormality and early pregnancy loss seen in patients with diabetes, particularly those with poor diabetic control.     

Human peripheral blood mononuclear cells (PBMC) in early pregnancy promote embryo invasion in vitro: HCG enhances the effects of PBMC.

BACKGROUND: The aim of this study was to investigate the role of peripheral blood mononuclear cells (PBMC) in embryo invasion at the implantation site and to estimate the effect on PBMC function of human chorionic gonadotrophin (HCG) that is secreted from the human embryo. METHODS AND RESULTS: The effect of PBMC on the invasiveness of murine embryos was examined using an invasion assay. PBMC obtained from women in early pregnancy (5-9 weeks gestation) significantly enhanced both spreading of murine embryos on Matrigel and invasion beneath the gel. These effects were greater than those of PBMC obtained from non-pregnant women in the secretory phase (cycle day 16-24) and the control (in the absence of PBMC). When PBMC obtained from non-pregnant women were incubated with recombinant HCG (10 IU/ml) for 2 days and were subjected to invasion assay using murine embryos, PBMC treated with HCG significantly promoted both spreading and invasion of murine embryos as compared with the non-treated PBMC. On the other hand, embryo outgrowth was not affected by HCG in the absence of PBMC, showing no direct effect of HCG on embryo invasion. CONCLUSION: This study indicated that PBMC from pregnant women promoted murine embryo invasion in vitro and this effect of PBMC was enhanced by HCG. These findings suggest that PBMC at the implantation site are activated by HCG secreted from the embryo, following which PBMC regulate embryo invasion.     

A prospective, randomized study comparing day 2 and day 3 embryo transfer in human IVF

It is believed that delayed transfer of embryos after IVF allows for a better selection of good quality embryos. Hence, the number of embryos and all other prognostic factors being equal, transfer of day 3 embryos should be associated with higher implantation and pregnancy rates than transfer of day 2 embryos. To investigate this hypothesis, a prospective randomized study was carried out to compare implantation and pregnancy rates between day 2 and day 3 transfers. The relationship between the embryo quality score of day 2 and day 3 embryos and their respective implantation rates was also analysed. In a 2 year period all patients undergoing infertility treatment and in whom at least seven normally fertilized oocytes were obtained were included in the study. A minimization procedure was performed taking into account the patient's age and the method of fertilization (IVF or intracytoplasmic sperm injection). By using a uniform policy of embryo transfer, the number of embryos transferred was similar in both groups. The outcome parameters were embryo quality, implantation and pregnancy rates. No difference was observed in implantation and pregnancy rates between transfers on day 2 versus day 3 (23.8 versus 23.8% and 47.9 versus 46.8% respectively). The incidence of embryos of moderate to poor quality was higher in embryos cultured for 3 days compared with those cultured for 2 days. It is concluded that the outcomes of embryo transfer in terms of implantation and pregnancy rates are comparable for day 2 and day 3 embryos, although the overall embryo quality score decreases when embryos are kept in culture till day 3.     

Development of a metabolic activation system for the frog embryo teratogenesis assay: Xenopus (FETAX)

FETAX (frog embryo teratogenesis assay: Xenopus) is a 96-hr teratogenesis screening assay using embryos of the South African clawed frog, Xenopus laevis. Since Xenopus embryos have limited xenobiotic metabolism through 96 hr of development, we have developed an in vitro metabolic activation system employing Aroclor 1254-induced rat liver microsomes. By adding an exogenous source of mixed functional oxidase (MFO) activity, we may more accurately assess the teratogenic risk of proteratogenic compounds. Xenopus embryos were cocultured with varying concentrations of cyclophosphamide (CP), Aroclor 1254-induced microsomal protein, an NADPH-generating system, and antibiotics in a static renewal fashion for 96 hr. Residual Aroclor 1254 remaining in the microsomes was successfully reduced during purification to levels that had no significant effect on embryo survival and development. The results of three definitive dose-response tests performed with CP revealed that activation reduced the 96 hr LC50 from 8.0 to 1.4 mg/ml (5.7-fold). The 96-hr EC50 (malformation) was reduced from 6.2 to 0.4 mg/ml (15.5-fold). Activation also increased the types and severity of malformation and reduced embryonic growth. Aroclor 1254-induced rat liver microsomes may be used as an acceptable in vitro metabolic activation system for FETAX.     

Supraphysiological estradiol levels do not affect oocyte and embryo quality in oocyte donation cycles.

BACKGROUND: The study aim was to determine whether supraphysiological estradiol (E(2)) levels reduce oocyte/embryo quality in oocyte donation cycles. METHODS: A retrospective analysis of 330 consecutive fresh oocyte donation cycles was performed in an assisted reproductive treatment programme between January 1996 and December 2000. Throughout the study period, oocyte donors and recipients followed a standard synchronization regimen that did not vary. A serum E(2) level (peak E(2)) was obtained from all oocyte donors on the morning of HCG administration. Peak E(2) values were grouped by 33rd percentile (group I, <1500 pg/ml; group II, 1500-3000 pg/ml; and group III, >3000 pg/ml). All embryo transfers were performed on day 3 after oocyte recovery. RESULTS: Comparisons between groups revealed no significant differences in the quality of oocytes retrieved, and in fertilization rates. Higher peak E(2) levels were directly correlated with a greater number of oocytes retrieved, embryos available for transfer and cryopreservation, and higher average embryo quality scores (P < 0.005). Compared with group I, group III had significantly higher embryo implantation rates (P < 0.05). CONCLUSIONS: Sustained supraphysiological E(2) levels do not adversely affect the quality of developing oocytes and embryos. On the contrary, elevated E(2) levels are associated with a larger number of oocytes and embryos and high-grade embryos for transfer/cryopreservation and, consequently, improved implantation rates.     

Block to development in cultured rat 1-cell embryos is overcome using medium HECM-1.

Rat pronuclear embryos were cultured in hamster embryo culture medium-1 (HECM-1) or a modified Krebs-Ringer bicarbonate solution (mKRB). Embryo cultures in HECM-1 were also challenged with low oxygen concentrations. In HECM-1, 57.9% (70/121) of the pronuclear embryos developed into the 4-cell stage after 48 h of culture. The rates of 8-cell, morula and blastocyst formation were 32.2% (39/121), 17.4% (21/121) and 9.9% (12/121), respectively. On the other hand, in mKRB, rat pronuclear embryos showed developmental blockages at the 2-cell and 4-cell stages, and never developed beyond the 4-cell stage. The rate of blastocyst formation under a low oxygen concentration was 20.1% (43/214), showing a significant difference from the value of 5.5% (11/201) obtained under a standard oxygen concentration (P less than 0.005). This is the first report of successful culture of rat pronuclear embryos to the blastocyst stage. Furthermore, it is suggested that protection from oxidation stress is a prerequisite for rat embryo development in vitro.