Culture Of Early Embryos Research Articles

Development retardation in cultured preimplantation rabbit embryos.

Day 3 to Day 5 preimplantation rabbit embryos were cultured for 24 h in chemically defined media which are widely used in early embryo culture (BSM II and Ham's F-10) supplemented with BSA or homologous serum. For the next 24 h, the embryos were left in the same culture medium, placed in freshly made medium, or cultured in medium which was supplemented with uterine flushings. In addition, 24-h cultured embryos were transferred into uteri of synchronous recipients for 1 day. After culture or transfer, development was assessed by cell proliferation evaluated by incorporation of tritiated thymidine. In comparison to non-cultured controls, thymidine incorporation demonstrated a considerably impaired cell proliferation after culture in defined media irrespective of medium, supplement, or replenishment with fresh medium. For Day 3 embryos, there was a developmental retardation amounting to about 1 day after 2 days in culture. Compared to Day 3 embryos, delay was clearly more pronounced in Day 4 and Day 5 blastocysts, i.e. in stages which had been retrieved from the uterus before culture. Supplementation with uterine flushings markedly promoted blastocyst cell proliferation (P less than 0.001). Incorporation data examined after transfer showed that impairment of cell proliferation caused by 1 day in culture had been compensated for to a large extent within 1 day in utero.

Chapter 10 Immunological Aspects of Implantation and Fetal Survival: The Central Role of Trophoblast

Publisher Summary This chapter discusses major histocompatibility complex (MHC) antigen expression on the developing embryo and trophoblast susceptibility to immune attack. Although there have been many studies on a variety of embryonic cells at different stages of gestation, it is relevant to consider only the trophectoderm of the blastocyst and its descendant trophoblast populations. Apart from certain extraembryonic membranes, trophoblast is the only fetal tissue in direct, unbroken, and continual contact with the maternal environment throughout gestation. During this time it undergoes extensive proliferation and differentiation, leading to the formation of biologically differing subpopulations in the mature placenta and placental bed. Current understanding of MHC antigen expression on mouse trophoblast has been derived from two different experimental approaches. The first involves the short-term culture of early embryos or placental cell suspensions in vitro. The second is the autoradiographic localization of the binding sites of radiolabeled monoclonal antibodies injected into the pregnant female. It demonstrates that MHC antigen expression on rat-placental trophoblast mirrors closely that seen in the mouse. The survival of the postimplantation embryo is ensured by the encapsulating barrier of trophoblastic cells and outer extraembryonic membranes that prevent the entry of maternal immunocompetent cells. The survival of the fetal barrier tissues themselves is primarily due to the failure of the pregnant female to mount a classical rejection response, possibly owing to the production of one or more immunomodulating factors by the trophoblast or soluble suppressor factors by maternal lymphocytes.

Modification and standardization of the culture of early postimplantation embryos for toxicological studies

The method of culturing "whole" rat embryos (days 9.5-11.5 of gestation, i.e. at the early stage of organogenesis) as modified and standardized in our laboratory is presented; We have succeeded in using bovine serum as culture medium instead of rat serum as recommended in the original procedure. Experimental conditions are described for obtaining reproducible results; An improved scoring system was developed which, in connection with a computerized documentation, greatly facilitates the evaluation of the data.

Fetal abnormality (Goldenhar syndrome) occurring in one of triplet infants derived from in vitro fertilization with possible monozygotic twinning.

Of 36 infants delivered following conception by in vitro fertilization (IVF), 1 case of significant fetal abnormality has been detected. The infant is one of male triplets exposed to medroxyprogesterone acetate (MPA) in utero and demonstrates abnormalities of the ear, vertebral column, and ribs which fit clearly into the oculoauriculovertebral syndrome described by Goldenhar and which have been reported in monozygotic twins with discordance. Marker studies including eight blood groups, HLA haplotype assignments, and banding studies of the chromosomes indicate that the infant and one of his brothers may well be identical (P less than 0.001 for dizygosity) except for the Fy(a) antigen. It is deduced that the fetal abnormality is not causally related either to MPA exposure or to the techniques applied during fertilization and early embryo culture, and furthermore, if the infant is the monozygotic twin of one of his brothers, the two phenomena are unrelated, as each was likely to have occurred at different stages of embryonic development.

Serum is not necessary in human in vitro fertilization, early embryo culture, and transfer

Human in vitro fertilization (IVF), embryo culture (EC), and embryo transfer (ET) are commonly performed in various media supplemented with blood serum. To determine whether serum is really necessary, the results of IVF, EC, and ET were compared by using two types of media: B2 medium supplemented with human cord serum and B3 medium without any serum. B3 medium is similar to B2, except that it contains 1% pure controlled human serum albumin in place of 1% bovine serum albumin. We did not observe any difference between the results obtained by using B2 or B3 at any phase of IVF, EC, and ET processes. Both media give an overall evolutive pregnancy rate of about 16%. B3 medium with serum seems to increase slightly the cleavage speed. Our results indicate that there are no positive effects when serum is used for human IVF-ET. To avoid serum supplementation is of evident interest for the homogeneity of the results. This will also lead to a better understanding of human early development and control of the egg quality by metabolic analysis of the media following in vitro EC.

In vitro culture of early bovine embryos with trophoblastic vesicles: Cleavage through the block stage, followed by pregnancy after transfer

To evaluate the direct effect of photodynamic treatment with 5-aminolevulinic acid (ALA) on preimplantation mouse embryos in an in vitro setting.Preimplantation mouse embryos were incubated with or without ALA for 5 hours and followed immediately by light exposure for 0, 5, or 15 minutes. Comparison of the viability and blastocyst formation was made among different treatment groups.A conventional laboratory setting with embryo culture facilities.Female CD1 mice were superovulated with pregnant mare serum gonadotropin and hCG before mating. Four- and eight-cell embryos and compacted morulae were flushed from the oviducts and incubated with 0, 0.1, 0.5, 1.0, or 5.0 mM ALA for 5 hours. Embryos subsequently were exposed to photoactivating light for 0, 5, or 15 minutes.Microscopic assessment of embryo quality at 12 hours and determination of the percentage of embryos progressing to the blastocyst stage at 36 or 60 hours.Incubation of embryos with 0.1, 0.5, 1.0, 5.0 mM ALA without light resulted in 87.3% ± 1.6%, 84.9% ± 3.4%, 81.4% ± 1.8%, and 82.8% ± 4.7% of the embryos developing to blastocysts, respectively. In the absence of ALA, light exposure for 0, 5, or 15 minutes resulted in 93.8% ± 2.3%, 92.3% ± 2.2%, and 85.9% ± 1.7% blastocyst formation. Combining treatment of ALA at the same concentrations with light resulted in 33.3% ± 2.1%, 0.7% ± 0.9%, 0%, 0% (5-minute light), 13.3% ± 1.0%, 0%, 1.6% ± 1.3%, 0% (15-minute light) blastocyst formation, respectively. When gross morphology was used to assess embryo viability at 12 hours, similar results were observed. Measurement of the fluorescent spectrum of embryos incubated with ALA indicated that protoporphyrin IX had been formed.Photodynamic ablation of mouse embryos was achieved with ALA under in vitro conditions. These results indicate that preimplantation mouse embryos are capable of converting ALA to the photosensitizer, protoporphyrin IX, and are susceptible to subsequent photoablation. A photodynamic effect on the embryo may be important to the successful application of this technique to the treatment of human ectopic pregnancy.

Culture of early somite mouse embryos during organogenesis

Early somite (2-4) mouse embryos were explanted and then maintained in culture for 24 or 48 h intervals. Various types of media were tested and it was determined that rat serum supported normal growth over a period of 48 h, based on total protein analysis and histological comparisons with in vivo specimens. Other media including fetal calf serum and fetal calf serum and Waymouth's (1:1) supported some growth, but did not equal the success of using rat serum alone. During the 48 h culture period in rat serum, embryos developed to stages indistinguishable from embryos maintained for a similar time in vivo.