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Biomedical subjects

B Behr

Publications and source records attributed to B Behr.

15 recordsLinked to original sources

Effects of culture conditions on IVF outcome.

Although in vitro fertilization (IVF) success rates have improved over the past decade, multiple pregnancies have become a formidable problem. The solution to this problem seems simple by mandating the reduction in numbers of embryos transferred. However, this is typically not accomplished without a compromise in the pregnancy rate. There have been a number of approaches designed to address high order multiple pregnancies from multi factorial analysis of early cleavage stage embryos to the development of extended culture systems, both of which require manipulations in the culture environment. Manipulations in embryo culture environment may not be benign. Several studies have demonstrated that adverse culture conditions have effects on gene expression and imprinting. Studies have also demonstrated that singleton human IVF babies have lower birth weight and higher incidence of congenital anomalies than natural conception babies. All of these factors need to be considered in relation to long term viability of IVF babies and the Barker hypothesis.

Culture Media↗

Vascular endothelial growth factor (VEGF) mRNA splice variants are differentially expressed in human blastocysts.

The aim of our study was to detect and characterize mRNA expression of VEGF isoforms VEGF(121), VEGF(145), VEGF(165), VEGF(189), and VEGF(206) in human blastocysts. We recently demonstrated VEGF mRNA expression during human preimplantation embryo development, and further information regarding the alternatively spliced mRNAs resulting in freely secreted proteins or proteins bound to cell surface heparan-sulphate proteoglycans is needed to better understand the process of angiogenesis during implantation. Human blastocysts unsuitable for transfer obtained from the IVF programme at Stanford University were examined by reverse transcription/hemi-nested polymerase chain reaction for their expression of VEGF mRNA splice variants. VEGF mRNA was expressed in 17 out of 19 (89%) blastocysts. Of the 17 blastocysts, VEGF(121) mRNA was detected in 88%, VEGF(145) mRNA in 100%, VEGF(165) mRNA in 71%, and VEGF(189) mRNA in 24% of blastocysts. There was co-expression of mRNA for VEGF(121) and VEGF(145) only in 29% blastocysts, of mRNA for VEGF(165) and VEGF(145) only in 12%, and of mRNA for VEGF(121), VEGF(145) and VEGF(165) in 59% blastocysts. VEGF(206) mRNA could not be detected. In conclusion, we demonstrated that blastocysts express the mRNAs encoding for the free VEGF proteins, enabling the implanting embryo to immediately induce angiogenesis at the implantation site.

Actins↗

Expression of vascular endothelial growth factor mRNA in human preimplantation embryos derived from tripronuclear zygotes.

OBJECTIVE: To detect the expression of vascular endothelial growth factor (VEGF) mRNA and/or secretion of VEGF protein by human preimplantation embryos. DESIGN: Human preimplantation embryos not suitable for uterine transfer were examined for beta-actin and VEGF mRNA expression. Culture media from normally fertilized and developing preimplantation embryos were assessed for VEGF protein secretion. SETTING: Clinics and academic research laboratories at the Departments of Obstetrics and Gynecology at the Stanford University, Palo Alto, California and the Heinrich-Heine-University, Düsseldorf, Germany. PATIENT(S): Couples undergoing IVF by intracytoplasmic sperm injection for various reasons. INTERVENTION(S): Six unfertilized oocytes and 33 pathologically fertilized (tripronucleic, 3PN) preimplantation embryos were examined for VEGF mRNA expression, and 16 embryos were examined for VEGF protein secretion. MAIN OUTCOME MEASURE(S): Embryonic expression of VEGF mRNA and VEGF protein as determined by reverse transcription (RT)/nested polymerase chain reaction (PCR) and ELISA. RESULT(S): VEGF mRNA and protein could not be detected in unfertilized oocytes. However, 30/33 preimplantation embryos did express VEGF mRNA (11/12 10-to-16-cell embryos, 3/4 morulae, 11/12 early blastocysts, 5/5 hatched blastocysts). The VEGF protein level was below the sensitivity of the ELISA. CONCLUSION(S): Production of VEGF may give the embryo the ability to induce neoangiogenesis at the implantation site, thus creating an environment necessary for its survival.

Cell Nucleus↗

Comparison of blastocyst transfer with day 3 embryo transfer in similar patient populations.

OBJECTIVE: To compare implantation and pregnancy rates (PRs) achieved with blastocyst transfer (BT) and day 3 ET in similar patient populations. DESIGN: Retrospective analysis. SETTING: Academic infertility center. PATIENT(S): One hundred consecutive patients <40 years undergoing IVF, each with more than three eight-cell embryos on day 3. INTERVENTION(S): Patients used their own eggs for IVF or IVF and intracytoplasmic sperm injection. Embryos were cultured in P1 medium (Irvine Scientific, Santa Ana, CA) until day 3, when they were either transferred or, in the case of embryos for BT, incubated in Blastocyst Medium (Irvine Scientific), followed by transferring on day 5. MAIN OUTCOME MEASURE(S): Implantation and PRs. RESULT(S): There were no statistically significant differences in patient age, FSH level, or number of oocytes or zygotes. The BT group had fewer embryos transferred (mean, 2.4) compared with the day 3-ET group (mean, 4.6). The viable PR (cardiac activity at 6-7 weeks was considered indicative of a viable pregnancy) was higher with BT (68%, 34/50) than with day 3 ET (46%, 23/50). The implantation rate was increased with BT (47%, 56 sacs/120 embryos) compared with day 3 ET (20%, 46 sacs/231 embryos). CONCLUSION(S): The BT group in our study had higher implantation and PRs compared with the day 3-ET group. Better embryo selection, improved embryo-uterine synchrony, and decreased cervical mucus on day 5 may have accounted for the enhanced outcome. Our data support the use of BT to limit the number of embryos transferred while improving PRs.

Adult↗

Blastocyst-ET and monozygotic twinning.

PURPOSE: To examine the rate of monozygotic twinning associated with blastocyst transfer using commercially available, cell-free culture systems with unmanipulated blastocysts. METHODS: A retrospective analysis was conducted in multiple private and academic infertility centers throughout the United States, of 199 pregnant patients following in vitro fertilization (IVF) blastocyst embryo transfer (ET). Human embryos obtained through standard IVF stimulation protocols were cultured in commercially available, cell-free media systems and transferred as blastocysts. The main outcome measure was the rate of monozygotic twinning. RESULTS: A total of 199 blastocyst-ET pregnancies were achieved during the study period at the fertility centers examined. Monozygotic twinning was noted in 10/199 (5%) of these pregnancies. All were monochorionic diamnionic. CONCLUSIONS: Monozygotic twinning previously has been reported following IVF, especially in relation to assisted hatching. While blastocyst transfer has been available for many years using coculture, there have been no published multicenter reports of monozygotic twinning associated with unmanipulated blastocysts. In a multicenter analysis, a definite increase in monozygotic twinning was seen following blastocyst-ET. We believe this phenomenon is real and that this information should be considered when counseling patients for treatment.

Cell-Free System↗

[Culture of blastocysts using sequential media].

The advantages of cultivating human pre-embryos to the stage of blastocites, are well known. The use of media of culture sequential, without the backing of somatic cells, is new. The objective was to cultivate human pre-embryos to the stage of blastocyte to the determine the recuperation indexes, implantation index and pregnancy index in patients subjected to FIVT/TE or ICSI. Once obtained the ovules of patients were injected/inseminated to latter be cultivated for 72 hours using P1 medium at 10% of SSS under mineral oil for, latter, be transferred to a complex medium for blastocites culture for 48 hours, and finally be transferred. Thirteen were included (9 of FIV and 4 of ICSI) obtaining 205 ovules; fertilized 143 and 131 had cellular division. One hundred and twenty one pre-embryos were cultivated to blastocyst stage, from which, 53 reached that stage (43.8%); transferring 28 and freezing 25. In average, 2.1 blastocysts were transferred by patient. There were four pregnancies and one alive newborn, at term for an index of implantation of 14.2% and a pregnancy index of 30.7%. The study shows our initial experience, which demonstrated and acceptable idea of recuperation of blastocysts and pregnancies.

Blastocyst↗

Sibling embryo blastocyst development correlates with the in vitro fertilization day 3 embryo transfer pregnancy rate in patients under age 40.

OBJECTIVE: To examine the IVF day 3-ET pregnancy rate in patients under 40 with sibling embryo blastocyst development, compared with similar patients without blastocyst formation. DESIGN: Retrospective analysis. SETTING: Academic infertility center. PATIENT(S): One hundred twenty-five IVF day 3-ET patients under 40 with sibling embryos for extended culture. INTERVENTION(S): Extended culture of nontransferred sibling embryos for blastocyst development. MAIN OUTCOME MEASURE(S): Pregnancy and multiple gestation rates, number of oocytes, embryos formed, and embryos transferred. RESULT(S): Thirty-eight percent of patients became pregnant. Forty-eight percent of patients had sibling embryos develop to blastocyst. The blastocyst group had more oocytes retrieved (17.4+/-6.6 versus 14.4+/-5.6), more embryos formed (11.2+/-4.2 versus 8.8+/-3.2), and a higher clinical pregnancy rate (60% versus 18%) than the group without blastocyst development. CONCLUSION(S): Blastocyst transfer has been shown to improve implantation rates and reduce the risk of multiple gestations from assisted reproductive technology. Sibling embryo blastocyst development may reflect superior embryo quality, as manifested by increased IVF-ET pregnancy rates. In addition to predicting pregnancy in the current cycle, sibling embryo blastocyst development may provide information about the potential for fresh blastocyst transfer in subsequent cycles and help to identify patients at risk for multiple gestations.

Adult↗

Two-blastocyst transfer has similar pregnancy rates and a decreased multiple gestation rate compared with three-blastocyst transfer.

OBJECTIVE: To examine the effect of the number of blastocysts transferred on pregnancy and multiple gestation rates. DESIGN: Retrospective study. SETTING: Academic infertility center. PATIENT(S): Patients < 40 years undergoing IVF, with FSH levels of < 15 mIU/mL and more than three eight-cell embryos. INTERVENTION(S): Embryos were cultured in P1 until day 3 and then transferred to blastocyst medium. A maximum of three blastocysts were transferred. MAIN OUTCOME MEASURE(S): Pregnancy, multiple gestation, and implantation rates. RESULT(S): All 55 patients developed blastocysts and underwent ET. Twenty-four patients had three embryos transferred and 29 patients had two embryos transferred. Two patients had only one embryo each for transfer. There was no difference in the viable pregnancy rate between the two-blastocyst transfer and three-blastocyst transfer groups (62% vs. 58%). In the two-blastocyst transfer group, 39% of pregnancies were multiple gestations (all twin gestations), compared with 79% of pregnancies in the three-blastocyst transfer group (50% twin gestations, 29% triplet gestations). The implantation rate was 47% in both groups. CONCLUSION(S): A commercially available, sequential culture system is highly effective for producing viable blastocysts. Two-blastocyst transfer eliminated the risk of triplets while maintaining the same high success rates seen with three-blastocyst ET.

Adult↗

Preliminary clinical experience with human blastocyst development in vitro without co-culture.

This preliminary analysis was designed to quantify blastocyst development of supernumerary embryos without the use of feeder cells, conditioned medium or whole serum. Embryos derived from in-vitro fertilization (IVF) that were not transferred or cryopreserved were included in this study. Ova were harvested for IVF after a standard ovarian stimulation with gonadotrophin-releasing hormone agonist/ human menopausal gonadotrophin (GnRHa/HMG) or follicle-stimulating hormone (FSH). Ova were collected and culture in 150 microliters droplets of P1 medium under mineral oil, in groups at 37 degrees C under 5% CO2, 5% O2, 90% N2 (group A) or under 5% CO2 in air (group B) environment. Embryo transfer was performed 72 h post-harvest. Viable embryos not transferred or cryopreserved were placed in blastocyst medium and cultured for an additional 48 h in 5% CO2 in air. Embryos that exhibited an expanded blastocoelic cavity and well-defined inner cell mass at 120 h were counted. Of 838 supernumerary embryos cultured, 448 (53.5%) reached the expanded blastocyst stage by 120 h of culture. Patients were given the option of cryopreservation at that time. The embryos were cryopreserved using a standard protocol with serial addition of glycerol. Embryos reaching the blastocyst stage after more than 120 h of culture were not included. There was no difference in the proportions of blastocyst development between group A, 217/410 (53.5%) and group B, 231/428 (54%). To date, 16 patients have each had up to three thawed blastocysts transferred, out of whom seven became pregnant. This report demonstrates that a simple system of sequential culture generated acceptable, viable blastocyst development (54%) with supernumerary embryos, without the use of feeder cells, conditioned medium or whole serum. Recognizing the differential metabolic requirements of early and late cleavage stage embryos has enabled the application of a glucose/phosphate-free simple culture medium (P1) for up to 72 h of culture and a complex, glucose-containing medium (blastocyst medium) for subsequent blastocyst development.

Blastocyst↗

Enhancement of motility and acrosome reaction in human spermatozoa: differential activation by type-specific phosphodiesterase inhibitors.

Inhibition of sperm phosphodiesterase (PDE) has been shown to increase cAMP concentrations and stimulate motility and the acrosome reaction. While several PDE genes exist in mammals, little is known about the physiological role of PDE forms expressed in human spermatozoa. Using type-selective inhibitors, we identified two of the PDE forms expressed in human spermatozoa and studied their involvement in sperm function. Selective inhibitors of calcium-calmodulin-regulated PDE1 (8-methoxy-isobutyl-methylxanthine) and cAMP-specific PDE4 (RS-25344, Rolipram) were used to study PDE forms in human sperm extracts. 8-MeIBMX and Rolipram/RS-25344 inhibited sperm PDE activity by 35-40 and 25-30% respectively. Subcellular fractionation of the sperm homogenate suggests these pharmacologically distinct forms may be located in separate cellular regions. To evaluate the functional significance of different PDE forms, the effect of type-specific PDE inhibition on sperm motility and the acrosome reaction was examined. PDE4 inhibitors enhanced sperm motility over controls without affecting the acrosome reaction, while PDE1 inhibitors selectively stimulated the acrosome reaction. These data indicate at least two distinct PDE types exist in human spermatozoa. Our findings also support the hypothesis that PDE subtypes affect sperm function by regulating separate pools of cAMP and may ultimately offer novel treatments to infertile couples with abnormal semen parameters.

1-Methyl-3-isobutylxanthine↗

Single blastomeres within human preimplantation embryos express different amounts of messenger ribonucleic acid for beta-actin and interleukin-1 receptor type I.

Gaining knowledge about the physiological timetable of gene expression during preimplantation embryo development is crucial, and a better understanding of cytokine and growth factor expression in early embryonic development could lead to improved in vitro culture conditions and enhance in vitro fertilization implantation rates. Our aim was to detect the patterns and levels of two messenger ribonucleic acids [mRNAs; beta-actin and interleukin-1 receptor type I (IL-1R tI)] in single human blastomeres by RT-nested PCR and to compare possible variations in the gene expression both between different embryos and in multiple blastomeres within the same embryo. Single blastomeres from nine human tripronucleic preimplantation embryos were examined by one round of RT and two rounds of nested competitive PCR. Beta-actin mRNA was detected in each blastomere, and IL-1R tI mRNA was found in 72% of the blastomeres examined. Beta-actin was expressed at a level of 511-12185 molecules of complementary DNA/blastomere, and IL-1R tI was expressed at a level of 2-290 molecules of complementary DNA/blastomere. Our results suggest that the mRNA pattern of an embryo cannot be reliably quantitated from the mRNA pattern of a single blastomere and therefore imply limitations for the use of this method for preimplantation diagnosis.

Actins↗

Genetic expression of hexokinase and glucose phosphate isomerase in late-stage mouse preimplantation embryos: transcription activities in glucose/phosphate-containing HTF and glucose/phosphate-free P1 media.

In mouse and human preimplantation development, pyruvate is consumed preferentially during early embryogenesis; however, during the morula and blastocyst stages, glucose is the preferred energy substrate. Studies have suggested that the glycolytic enzymes, hexokinase and glucose phosphate isomerase, are important enzymes in glucose metabolism during these later stages of human and mouse preimplantation development. In order to investigate the genetic activities of these enzymes in late-stage mouse embryos developing in vitro, we analysed hexokinase and glucose phosphate isomerase transcription activities by qualitative RNA assays using reverse transcriptase-nested polymerase chain reaction amplification of individual mouse morulae and early blastocysts incubated in glucose/phosphate-free preimplantation stage one (P1) medium and glucose/phosphate-containing human tubal fluid (HTF) medium. We observed an increased incidence of hexokinase transcripts in the population of blastocysts compared with morulae, and differences in transcript incidence between early blastocysts developing in HTF medium and in P1 medium. In contrast, glucose phosphate isomerase transcripts were consistantly present in all embryos analysed, and appear to be constitutively expressed during late-stage mouse embryogenesis. The different activity patterns of the two glycolytic genes may reflect different mechanisms of gene regulation or differential transcript stability during the later stages of mouse preimplantation development.

Animals↗

Quantification of hexokinase mRNA in mouse blastocysts by competitive reverse transcriptase polymerase chain reaction.

Hexokinase (HX), the enzyme that catalyses the initial reaction in glycolysis, is an important enzyme in glucose metabolism during human and mouse embryonic development. In our previous investigations of the genetic activities of HX, we observed an increased incidence of HX gene expression in blastocysts in comparison with morulae, and variability in the incidence of HX gene expression between embryos at the same developmental stages. These observations prompted us to quantify HX mRNA in mouse blastocysts to define the biological significance of the variable gene transcription. We modified our qualitative reverse transcription-nested polymerase chain reaction (RT-nPCR) assay for HX mRNA in single or groups of embryos to quantify HX mRNA by competitive RT-nPCR. HX mRNA was quantified in cohorts of mouse blastocysts cultured in glucose/phosphate-containing human tubal fluid (HTF) media. These blastocysts expressed HX in minute amounts, averaging 1.95 x 10(-18) g of mRNA. This is the first attempt at quantification of single gene mRNA in preimplantation embryos. Further investigations using similar techniques will enable comparative analyses between embryos to be performed to determine the correlation between specific levels of HX mRNA transcripts in individual embryos and embryonic viability and competence for further development and implantation.

Animals↗

Factors related to early pregnancy recognition in the woman.

PURPOSE: While the precise timing of the maternal recognition of pregnancy is not known, it is known that the prevention of return to ovarian cyclicity relies on a conceptus-derived signal. METHODS: In an attempt to identify the first luteotropic signals detectable in the maternal circulation, a sensitive Leydig cell luteotropin bioassay was employed, and data were compared for nine clinically pregnant and nine nonpregnant patients in an in vitro fertilization program. Blood samples were drawn every other day for 10 days after embryo transfer (ET). RESULTS: The first detectable rise in bioactive luteotropin levels occurred between 6 and 8 days post ET. Serum E2 levels increased on the same days. Differences in luteotropin levels between pregnant and nonpregnant patients were significant between days 6 and 8 (P < 0.0001) and between days 8 and 10 (P < 0.002). CONCLUSIONS: Based on morphological studies reported by others, bioactive luteotropic signals identified in this study were detectable in the maternal circulation at about the time of trophoblast lacunae coalescing with maternal uterine blood vessels.

Animals↗