A simplified technique for fertilization and culture of human preimplantation embryos in vitro

The paper describes simplification of existing methods of culture in human IVF. Focusing attention on the need to streamline procedures, the mode of housing the egg/embryo in a constant accessible environment is examined. We now culture with B2/B3-Menezo medium in a multidish under 5% CO2 in air at 37 degrees C in an open system within a CO2 gassing incubator. The advent of the multidish as a specific housing system is highlighted. The 80% fertilization rate combined with a stable osmolarity demonstrates that previous success is maintained with changes introduced to improve convenience and simplicity of procedures.     

Growth of human preimplantation embryos in vitro

The human oocyte fertilizes and develops into embryos in the Fallopian tube and reaches the uterus only after compaction. However, for several years embryos that were developed following in-vitro fertilization (IVF) were transferred into the uterus on day 2 or 3 at the 4-8 cell stage in contrast to the in-vivo situation where they would be present in the Fallopian tube. Earlier attempts to grow embryos in vitro for 5 to 6 days were not always successful. Attempts were therefore made to understand the in-vivo environment of the Fallopian tube where the early embryonic development occurs. This article reviews the studies carried out to understand the composition of fluids in the Fallopian tube specifically with reference to the energy metabolites - lactate, pyruvate and glucose; it also covers how the formulation of culture media for human IVF and embryonic development were modified over the years based on some classical work done on embryo culture in laboratory animals.     

A potential use of embryonic stem cell medium for the in vitro culture of preimplantation embryos

Purpose  

To assess the impact of embryonic stem cell culture medium (ESCM) on the pre- and post-implantation development of the mouse embryo, as a mammalian model, in comparison with the conventional culture medium, a potassium simplex optimized medium (KSOM).     

Expression of indoleamine 2,3-dioxygenase in the rhesus monkey and common marmoset

Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial and rate-limiting step of tryptophan degradation along the kynurenine pathway, and is hypothesized to limit tryptophan availability at embryo implantation and prevent maternal T cell activation at the maternal-fetal interface. To determine if nonhuman primates are suitable models for investigating the role of IDO during pregnancy, we defined the expression of IDO in the rhesus monkey and common marmoset with particular attention to the female reproductive tract and placenta. IDO mRNA was detected by RT-PCR in the rhesus monkey term placenta, lung, small intestine, spleen, lymph node and nonpregnant uterus, and also in the common marmoset placenta. Immunohistochemical analysis of rhesus monkey tissues localized IDO to glandular epithelium of nonpregnant endometrium and first trimester decidua, vessel endothelium of nonpregnant myometrium, first trimester decidua and term decidua, and villous vessel endothelium and syncytiotrophoblast of term placenta. Western blot analysis confirmed IDO in rhesus monkey term placenta. In the common marmoset, IDO was detected in glandular epithelium of the nonpregnant uterus and in the decidua at day 60 and day 128 of gestation. IDO activity was higher in rhesus monkey and common marmoset decidua and placentas than in other tissues. Confirmation of IDO expression in rhesus monkey and common marmoset uterine and placental tissues supports the hypothesis that this enzyme regulates immune activation at the maternal-fetal interface and demonstrates that nonhuman primates may provide models with distinct similarities to human placentation to study the role of IDO in maternal-fetal immune dialogue.     

Expression of indoleamine 2,3-dioxygenase in the rhesus monkey and common marmoset

Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial and rate-limiting step of tryptophan degradation along the kynurenine pathway, and is hypothesized to limit tryptophan availability at embryo implantation and prevent maternal T cell activation at the maternal–fetal interface. To determine if nonhuman primates are suitable models for investigating the role of IDO during pregnancy, we defined the expression of IDO in the rhesus monkey and common marmoset with particular attention to the female reproductive tract and placenta. IDO mRNA was detected by RT-PCR in the rhesus monkey term placenta, lung, small intestine, spleen, lymph node and nonpregnant uterus, and also in the common marmoset placenta. Immunohistochemical analysis of rhesus monkey tissues localized IDO to glandular epithelium of nonpregnant endometrium and first trimester decidua, vessel endothelium of nonpregnant myometrium, first trimester decidua and term decidua, and villous vessel endothelium and syncytiotrophoblast of term placenta. Western blot analysis confirmed IDO in rhesus monkey term placenta. In the common marmoset, IDO was detected in glandular epithelium of the nonpregnant uterus and in the decidua at day 60 and day 128 of gestation. IDO activity was higher in rhesus monkey and common marmoset decidua and placentas than in other tissues. Confirmation of IDO expression in rhesus monkey and common marmoset uterine and placental tissues supports the hypothesis that this enzyme regulates immune activation at the maternal–fetal interface and demonstrates that nonhuman primates may provide models with distinct similarities to human placentation to study the role of IDO in maternal–fetal immune dialogue.     

Births following the transfer of cultured embryos obtained by in vitro and in vivo fertilization in the marmoset monkey (Callithrix jacchus)

The aim of this study was to determine whether marmoset monkeys are a suitable primate model for in vitro fertilization (IVF), embryo culture, and transplantation studies. A prostaglandin analogue given in early pregnancy and human chorionic gonadotropin given near the end of the ensuing follicular phase were used for controlling the reproductive cycle, timing oocyte collection, and synchronizing the cycles of oocyte donors and embryo recipients. Five embryos obtained by IVF were transferred at early stages to the uterus of three recipients, and two gave birth to live infants. Some of the embryos were cultured to advanced blastocyst stages. In vivo fertilized oocytes were also cultured and transferred to two recipients, and one gave birth. We concluded that the marmoset is one of the best primates for such investigations.     

Enhanced effect of glycyl-L-glutamine on mouse preimplantation embryos in vitro

A comparison of the effects of replacing L -glutamine with either glycyl-L-glutamine or alanyl-L-glutamine in a KSOM-type medium on the development of mouse preimplantation embryos in vitro has been made. Alanyl-L-glutamine has no significant effect on the rates of blastocyst formation, onset or completion of hatching, and on the numbers of inner cell mass and trophectoderm cells that develop. Glycyl-L-glutamine has no effect on the rate of blastocyst formation; it stimulates slightly the onset of hatching, but significantly increases the numbers of inner cell mass and trophectoderm cells that develop. Embryo transfer experiments comparing media containing either glutamine or glycyl-L-glutamine have not produced any gross abnormal fetal development. Recently, alanyl-L-glutamine has been used to replace glutamine in media for the culture of human preimplantation embryos. The results in this paper suggest that glycyl-L-glutamine may be a better choice of dipeptide.     

Effect of anordrin on the development of mouse preimplantation embryos in vitro

Purpose: The in vitro effect of anordrin and anordiol on the development of mouse two-cell embryos was studied. Method: Female mice were primed with gonadotropins for superovulation and caged with male mice. Preimplantation embryos, at the two-cell stage, were recovered from the oviducts at 40 hr post-hCG. In the first experiment, two-cell embryos were exposed to culture medium containing different concentrations of anordrin for 3, 12, 24, and 80 hr and then grown in the anordrin-free culture medium and assessed for the formation of total and hatching blastocysts at 80 hr. In the second experiment, two-cell embryos were grown in culture medium containing different concentrations of anordiol and assessed for the formation of total and hatching blastocysts at 80 hr in vitro. Results: Exposure of two-cell embryos to anordrin concentrations of 2.5–7.5µg/ml for 12 hr, 2.5–5.0µg/ml for 24 hr, and 2.5µg/ml for 80 hr caused significant inhibition of the formation of total blastocysts and to 2.5–7.5µg/ml for 12 hr, 1.0–2.5µg/ml for 24 hr, and 1.0µg/ml for 80 hr caused significant inhibition of the formation of hatching blastocysts, in a exposure time-dependent and dose-dependent manner. Exposure of two-cell embryos to anordiol concentrations of 15–25µg/ml for 80 hr caused significant inhibition of the formation of total blastocysts and to 15–20µg/ml for 80 hr caused significant inhibition of the formation of hatching blastocysts in a dose-dependent manner. Conclusion: Anordrin and its metabolite anordiol inhibit the development of two-cell embryos in vitro.Presented orally at the 50th Annual Meeting of the American Fertility Society, San Antonio, Texas, November 5–10, 1994, Abstract No. O-176.     

Effects of immunoglobulins on in vitro hatching of preimplantation rabbit embryos

The following experiments were designed to study the effects of nonspecific immunoglobulins on the growth and development of the preimplantation rabbit embryo during in vitro cultivation. Embryos (2–4 cells) were cultured in the presence of nonspecific sheep serum gamma-globulins (SSGG) for 120 h. Growth and development of embryos were unaffected by SSGG (1.0 mg/ml) as 88% of the ova advanced to the blastocyst stage compared to 90% of the controls. Of the developed embryos, the percentages of late blastocysts with expanding blastocoels in the control and SSGG-treated cultures were 77% and 74%, respectively. However, the ability of the expanding embryo to shed its zona pellucida (ZP) was inhibited by the presence of SSGG. Of the total number of embryos cultured in control medium, 51% hatched from the ZP with 27% completely hatched. In the presence of SSGG (1.0 mg/ml) hatching (32%) was significantly inhibited (P<0.01) with no embryo completely hatched (P<0.005). This effect was not observed when embryos were cultured in 0.10 or 0.01 mg/ml of SSGG, suggesting a threshold of inhibition. Purified immunoglobulins from DEAE-cellulose chromatography had opposite effects on hatching depending on the net charge of the gamma-globulin. The more positive (acidic) immunoglobulin (IgG) molecules were unable to prevent in vitro hatching, while the more negative (basic) gamma-globulins significantly inhibited hatching. The immunoglobulin effect on embryonic hatching was independent of the species of origin since both sheep and rabbit gamma-globulins had similar results. This evidence suggests that immunoglobulins with a more negative net charge may inhibit embryonic proteolytic activity necessary for dissolution of the ZP which precedes in vitro hatching of expanded rabbit blastocysts.     

Acute effects of mercuric compounds on preimplantation mouse embryos in vitro

The effects of mercuric compounds on the proliferation and protein synthesis of mouse blastocysts were examined in vitro by treating the embryos with methylmercury (MMC) or mercuric chloride (MC). Late blastocysts were exposed to various concentrations of mercuric compounds for 24 hr. and incubated for another 24 hr. in a mercury-free medium. The protein synthesis of the mercury-treated blastocysts was measured by counting the incorporation of L-[35S]methionine into the acid-insoluble protein fraction of a cell. 0.1 microM of MMC was equivalent to 20 microM of MC with regard to the inhibitory effect on proliferation, and equivalent to 2 to 5 microM of MC after 24 hours' culture, and 10 to 20 microM after 48 hours' culture with regard to the inhibitory effect on protein synthesis of the blastocysts. The results showed that MMC was approximately 200 times more toxic than MC with regard to cell number proliferation, and 20 to 50 times so with regard to protein synthesis capability.     

The effects of serum from women with miscarriages on the in vitro development of mouse preimplantation embryos

It is generally believed that some human miscarriages result from embryotoxic factors existing in the sera. To study the embryotoxicity of such sera, 3.5-day-old mouse blastocysts were cultured for 72 h on 80% sera from different groups of women. After 72 h there was no blastocystic development in 53.2% of the cases grown on sera from women after two or more miscarriages, and none in 33.6% of the blastocysts grown on sera from women after one miscarriage, as compared with 8.2% and 12% respectively on control sera. Sera from women with miscarriages were divided into 'high risk' (50% or more embryotoxicity) and 'low risk' (less than 50% embryotoxicity) sera. The 'high risk' sera from two or more miscarriages caused an average of 72.1% undevelopment, while the 'low risk' sera (less than 50% embryotoxicity) from the same group caused 33.6% undevelopment. The 'high risk' sera from one miscarriage were embryotoxic to 55.8% of the blastocysts and the 'low risk' sera from the same group caused only 8.7% undevelopment. No significant differences were found in the mean serum concentrations of folic acid, zinc and copper of many of the experimental groups, in comparison with controls. The embryotoxic factor/s which exist in the 'high risk' sera from women with miscarriages are still not known.     

The effect of activin-a on the development of mouse preimplantation embryos in vitro

Purpose: Our purpose was to clarify the involvement of transforming growth factor- (TGF-) family in the regulation of preimplantation embryo development. Methods: The effects of activin-A and TGF- on the rates of morula and blastocyst formations as well as on the cleavage velocity of a mouse two-cell embryo in vitro were analyzed. The gene expressions of these two growth factors in various developmental stages were also studied using RT-PCR. Results: Activin-A at a concentration of 0.2 ng/ml significantly stimulated not only the rate of morula formation but also the velocity of embryo cleavage, whereas no significant effect was found with TGF-. RT-PCR revealed that activin-A subunit mRNA, but not TGF- mRNA, was detected in preimplantation mouse embryo at any developmental stage. Conclusions: Activin-A plays an important role in the regulation of preimplantation mouse embryo development in an autocrine fashion.     

Effect of chromosomal translocations on the development of preimplantation human embryos in vitro

OBJECTIVE: To determine the reliability of a new technique for single human blastomere karyotyping during clinical cases for preimplantation genetic diagnosis of translocations. DESIGN: Controlled clinical study. SETTING: Preimplantation genetic diagnosis and IVF program. PATIENT(S): Nineteen preimplantation genetic diagnosis cases with 11 types of translocations (10 reciprocal and one Robertsonian) involving chromosomes 1, 5, 7, 8, 9, 11, 12, 13, 14, 15, 16, 18, 20, 21, and 22. INTERVENTION(S): Blastomere biopsy followed by blastomere nucleus conversion into metaphase chromosomes. Fluorescent in situ hybridization (whole chromosome painting) was used for the detection of chromosomally unbalanced preimplantation human embryos.Main Outcome Measure(s): Percentage of informative metaphase plates and effect of unbalanced translocations on preimplantation embryo development. RESULT(S): Informative metaphases were obtained for 84% of the blastomeres. Analysis of preimplantation development of the resulting embryos showed that an unbalanced chromosomal complement does not affect embryo ability to reach the blastocyst stage in vitro. CONCLUSION(S): For the translocations tested, there is no evident selection against chromosomally unbalanced embryos at the preimplantation stage of embryo development.     

Chromosomal analysis of unfertilized oocytes and morphologically abnormal preimplantation embryos from an in vitro fertilization program

In vitro fertilization cycles yield a low percentage of pregnancies. Eighty-five to ninety percent of the transferred embryos do not implant, and the abortion rate approaches 30%. Aneuploidy is assumed to be responsible for a major portion of this pregnancy wastage. The purpose of this study was to determine if there was any correlation between morphology and chromosomal content of unfertilized oocytes and rejected embryos. To assess the chromosomal content of oocytes and embryos, we used the method described by Tarkowski in 1966. Sixty oocytes from 28 women, aged between 27 and 41 years, were analyzed. Sixty-seven percent were aneuploid; of these, 23.35% were hyperhaploid, 23.35% were hypohaploid, 8.35% were hyperdiploid, 3.35% were diploid, and 8.35% showed premature chromosome condensation. Of 20 preimplantation embryos analyzed, 80% were aneuploid, 10% were diploid, 5% were haploid, and 5% showed structural anomaly. Correlation was found between maternal age and aneuploidy in oocytes and between morphology and genetic balance in preimplantation embryos.     

Chromosomal information derived from single blastomeres isolated from cleavage-stage embryos and cultured in vitro

OBJECTIVE: To evaluate the potential of proliferation of single blastomeres isolated from human cleavage-stage embryos for use in preimplantation genetic diagnosis of chromosomal abnormalities. DESIGN: A laboratory study of chromosomal content of blastomeres isolated from embryos of patients from an in vitro fertilization program. SETTING: University hospital laboratory. PATIENT(S): Couples undergoing IVF or ICSI. INTERVENTION(S): Blastomeres were isolated from normally fertilized cleavage-stage human embryos, cultured in vitro or fixed immediately, and analyzed by fluorescence in situ hybridization (FISH) probes. MAIN OUTCOME MEASURES: Chromosomal information yielded by blastomeres cultured in vitro compared with those obtained from blastomeres that were processed for chromosomal analysis directly after isolation. RESULT(S): The percentage of cultured blastomeres that produced FISH results was significantly lower than the percentage of blastomeres processed for FISH directly after isolation (72% vs. 90%). Lack of FISH results from cultured cells, which in most cases was related to nuclear anomalies, was significantly more frequent among nondivided than divided blastomeres (39% vs. 21%). Both cultured and noncultured cells showed diploid, aneuploid and polyploid chromosome complements on FISH. Compared with directly processed cells, cultured cells yielded a higher proportion of polyploid patterns (22.9% vs. 6.1%). Of the cultured blastomeres that divided, 18% produced progeny with mosaicism. CONCLUSION(S): Although blastomere culture may increase the number of cells available for chromosomal analysis, the high frequency of nuclear defects and the occurrence of polyploidy and mosaicism among cultured cells discourage the use of blastomere isolation and proliferation strategy for use in preimplantation genetic diagnosis.