Oolemma rupture inside the intracytoplasmic sperm injection needle significantly improves the fertilization rate and reduces oocyte damage.

OBJECTIVE: To compare the effect on fertilization, oocyte damage, embryo freezing, and pregnancy rates of two different techniques for rupturing the oolemma during intracytoplasmic sperm injection (ICSI). DESIGN: Retrospective study. SETTING: Fertility Center, Alliant Health System Hospital. PATIENT(S): Seventy-nine consecutive IVF-ICSI cases. INTERVENTION(S): Patients in group I had ICSI performed by pushing the needle into the oocyte until the oolemma was observed to break outside the needle. In group II the oolemma was aspirated into the needle until it ruptured inside the needle. MAIN OUTCOME MEASURE(S): In group II ICSI resulted in significantly higher fertilization and lower oocyte damage rates (66% and 13%) than in group 1 (39% and 29%). There were no statistically significant differences in embryo cleavage rates or pregnancy rates per retrieval between the two groups. A greater number of cases had embryos cryopreserved in group II than in group I. RESULT(S): Rupturing the oolemma by aspirating it into the ICSI needle (group II) improved laboratory outcomes compared with the more traditional technique of breaking this membrane by the stabbing action of the needle (group I). This modification of the ICSI technique also increased the number of patients with cryopreserved embryos and therefore could increase the pregnancy rate per patient. CONCLUSION(S): The site and technique used to rupture the oolemma during ICSI has a significant effect on the fertilization and damage rates.

A simple approach to intracytoplasmic sperm injection.

OBJECTIVE: To describe a simple injection apparatus and method for performing intracytoplasmic sperm injection in a clinical IVF program. DESIGN: A prospective clinical trial of intracytoplasmic sperm injection. SETTING: A private office-based fertility program. PATIENTS: Five couples undergoing IVF-ET with intracytoplasmic sperm injection as a treatment for male factor infertility. INTERVENTIONS: Intracytoplasmic sperm injection was performed at room temperature (23.5 to 24.5 degrees C) in a simple zwitterion-buffered medium. MAIN OUTCOME MEASURES: Fertilization rates, cleavage rates, clinical pregnancy rates, implantation rates. RESULTS: Intracytoplasmic sperm injection was performed on 44 fresh oocytes from five patients. Twenty-three oocytes fertilized (52.3%) and 22 zygotes cleaved (95.7%). Three of five patients became pregnant (60%), resulting in the live birth of one normal male infant, one continuing singleton pregnancy, and one continuing twin gestation (46XX, 46XY). The implantation rate was 23.5%. CONCLUSION: Intracytoplasmic sperm injection can be performed successfully in a simple medium at room temperature using commercially available microtools.

Zygote intrafallopian transfer as a treatment for nontubal infertility: a 2-year study.

Zygote intrafallopian transfer (ZIFT) was used as a treatment for long-standing nontubal infertility for a 2-year period. The overall clinical pregnancy rate for 114 tubal transfers was 40.4% with a delivery/ongoing rate of 34.2%. Concurrent use of in vitro fertilization and embryo transfer (IVF-ET) for tubal factor infertility gave significantly lower clinical pregnancy and delivery/ongoing rates (21.1% and 15.8%, respectively). The use of gamete intrafallopian transfer (GIFT) for nontubal infertility yielded a 32% clinical pregnancy rate and a 26% delivery rate for 53 transfers. Zygote intrafallopian transfer resulted in an implantation rate per zygote of 17% overall compared with 8.1% per embryo for IVF-ET and 11.2% per oocyte for GIFT. The transfer of three zygotes per patient gave the same clinical pregnancy rate as the transfer of four while reducing the incidence of multiple gestation from 19% to 7.8% per transfer. No significant decline in the clinical pregnancy or delivery rate was seen with ZIFT in women aged 25 through 39.

Zygote intrafallopian transfer with "donor rescue": a new option for severe male factor infertility.

Clinical pregnancies have been initiated by ZIFT using zygotes produced by reinsemination of oocytes with donor sperm ("donor rescue") after an initial 15- to 20-hour exposure to husband's sperm. A total of 54 oocytes from four couples experiencing failed fertilization by husband's sperm were reinseminated with donor sperm, resulting in 38 zygotes (70.4% fertilization). Four zygotes were transferred during ZIFT in each case and resulted in two (50%) continuing pregnancies. Additional zygotes from donor reinsemination were cryopreserved for each couple. Donor rescue expands the utility of ZIFT as a treatment for male factor infertility.

Relative contribution of promoter and intragenic sequences in the hormonal regulation of rat beta-casein transgenes.

In order to identify DNA sequences responsible for the regulation beta-casein gene expression, lines of transgenic mice bearing the entire rat beta-casein gene and two rat beta-casein promoter chloramphenicol acetyltransferase (CAT) fusion genes have been established. All three transgenes have been shown previously to be regulated in a tissue- and stage specific manner. To investigate the relative contribution of promoter and intragenic sequences in the hormonal regulation of the beta-casein gene, mammary explant cultures derived from these lines of mice have now been performed, and the effects of PRL and glucocorticoids on transgene as compared with endogenous beta-casein gene expression have been quantified. After the addition of PRL to cultures performed in the presence of insulin and glucocorticoids, a 25- to 40-fold induction of endogenous mouse beta-casein mRNA was observed after 48 hr. A comparable greater than 25-fold induction of transgene expression after PRL addition was observed in explant cultures derived from a line of mice expressing the entire rat beta-casein gene. In contrast, PRL addition elicited only a 1- to 4.5-fold increase in CAT activity in cultures derived from two lines of mice bearing casein-CAT fusion genes with either 524 or 2300 base pairs of 5'-flanking DNA. In the presence of insulin, glucocorticoid or PRL addition alone increased endogenous beta-casein gene expression 2- to 2.5-fold and 5- to 10-fold, respectively, but only a 1.2- to 2.5-fold induction of CAT activity was observed for each hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice.

Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat beta-casein gene with 3.5 kilobases (kb) of 5' and 3.0 kb of 3' DNA in transgenic mice (Lee et al., Nucleic Acids Res. 16:1027-1041, 1988). In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat beta-casein promoter-bacterial chloramphenicol acetyltransferase (cat) fusion genes have been established. Twenty and eight lines of transgenic mice carrying two fusion genes containing either 2.3 or 0.5 kb, respectively, of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A were identified, most of which transmitted the transgenes to their offspring in a Mendelian pattern. CAT activity was detected predominantly in the lactating mammary gland of female transgenic mice but not in the male mammary fat pad. A several-hundred-fold variation in the level of cat expression was observed in the mammary gland of different lines of mice, presumably due to the site of integration of the transgenes. CAT activity was increased in the mammary gland during development from virgin to midpregnancy and lactation. Unexpectedly, the casein-cat transgenes were also expressed in the thymus of different lines of both male and female mice, in some cases at levels equivalent to those observed in the mammary gland, and in contrast to the mammary gland, CAT activity was decreased during pregnancy and lactation in the thymus. Thus, 0.5 kb of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A are sufficient to target bacterial cat gene expression to the mammary gland of lactating mice.

Tissue-specific expression of the rat beta-casein gene in transgenic mice.

The rat beta-casein gene is a member of a small gene family, encoding the principal milk proteins. In order to understand the mechanisms by which its stage- and tissue-specific expression are regulated, initially, a 14 kb genomic clone containing the entire 7.5 kb rat beta-casein gene with 3.5 kb of 5' and 3.0 kb of 3' flanking DNA was microinjected into the germline of mice. Eight F0 transgenic mice were generated with copy numbers ranging from 1-10; five transmitted the transgene to their offspring in a Mendelian pattern. A specific RNase protection assay was developed to quantitate the level of expression of the rat beta-casein transgene as compared to the endogenous mouse beta-casein gene. Using this assay expression was demonstrated predominantly in the lactating mammary gland of transgenic mice at a level of 0.01-1% of the endogenous mouse beta-casein gene. The transgene employed the authentic transcription initiation site observed previously in the analogous rat beta-casein gene. In one line, a reduced level of expression of the transgene was also observed in the brain. The site of integration, therefore, plays an important role in influencing the level of expression of the transgene, but not its general pattern of tissue-specificity. The transgene appears to be developmentally-regulated in accordance with the endogenous mouse beta-casein gene. These lines of mice generated carrying the rat beta-casein transgene should provide useful models for studying the developmental and hormonal regulation of milk protein gene expression.