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Presence of natural suppressor cells in the chicken embryo spleen and the effect of virus infection of the embryo on suppressor cell activity.

Spleen cells but not the thymus or the bursa cells of chicken embryos suppressed the in vitro mitogenesis of spleen cells of adult syngeneic or allogeneic chickens. The natural suppressor cell activity of embryo spleen was present at embryonation day 16, reached peak levels at embryonation day 18 and disappeared at hatch. The embryo spleen cells did not by themselves respond to phytohemagglutinin stimulation in vitro. The suppressive effect of embryonic spleen cells on adult spleen cells was present when the embryonic cells were added at the time of or after initiation of the adult spleen mitogenic cultures. When the embryonic cells were added to the cultures of adult spleen cells after the blastogenic response of the adult cells had peaked, the embryonic cells inhibited the incorporation of the label into adult spleen cell blasts. The suppressive activity of the embryonic spleen cells was mediated by soluble suppressor product(s) secreted by these cells, and direct cell-to-cell contact between embryonic and adult spleen cells was not necessary for suppression to occur. Infection of embryos with turkey herpesvirus and Marek's disease virus reduced the suppressor cell activity of embryonic spleen, although substantial residual suppressor cell activity remained in virus-infected embryos. Several pathogenic or non-pathogenic isolates of infectious bursal disease virus did not appreciably alter the suppressor cell activity of embryonic spleen cells.

Animals↗

Passage of herpes simplex virus type 1 on chick embryo fibroblasts confers virulence for chick embryos.

The pathogenesis of chorioallantoic membrane (CAM) infection with herpes simplex virus 1 and 2 (HSV-1 and HSV-2) as well as chick embryo fibroblast (CEF) passaged HSV-1 was studied. It was found that HSV-2 is at least a million-fold more virulent than HSV-1 as measured by pfu/LD50 ratios for the embryo. Serial passage of HSV-1 in vitro on CEF cells selected for a virus (CEFP10) which, unlike its parental HSV-1 strain, is able to kill the embryo. The restriction endonuclease maps of CEFP10 and its parental strain are indistinguishable and the mechanism of the increased virulence of CEFP10 was demonstrated to be its enhanced replication in chick embryo cells both in vivo and in vitro. In contrast, despite its inferior replicative ability, HSV-2 was found to have a biologically important specific invasiveness function that is not simply related to overall viral replication. Finally, the ability to isolate HSV-1 (CEFP10) virulent for the chick embryo after passage in vitro illustrates that tissue culture passage of HSV in appropriate cells may actually increase virulence for the animal host.

Animals↗

Cryopreserved embryo transfers suggest that endometrial receptivity may contribute to reduced success rates of later developing embryos.

OBJECTIVE: To evaluate viability and implantation potential of cryopreserved blastocysts according to the day of blastocyst expansion and cryopreservation. DESIGN: Retrospective study. SETTING: Private ART center. PATIENT(S): Three hundred and seventy-five patients undergoing embryo transfer with cryopreserved blastocysts. INTERVENTION(S): Blastocyst cryopreservation on day 5, 6, or 7 after oocyte retrieval according to the day of blastocyst expansion and subsequent embryo transfer. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate (PR) per embryo transfer. RESULT(S): Clinical PRs were similar between blastocysts cryopreserved on day 5 and blastocysts cryopreserved on day 6 (32% vs. 28%). The clinical PR was lower for blastocysts cryopreserved on day 7 (15%), but this difference was not statistically significant after accounting for the number of embryos per transfer (P=.15). CONCLUSION(S): Viability and implantation potential are similar for day 5 and day 6 blastocyst cryopreservation. Viability may be reduced for blastocysts cryopreserved on day 7, but not to the extent suggested by reports of fresh transfers. These results suggest that reduced success rates associated with fresh transfers of later developing blastocysts may be the result of asynchrony with endometrial receptivity instead of poorer embryo quality.

Adult↗

Establishment of an animal embryo culture system containing various embryotropins and its efficacy for culturing ICR mouse one-cell embryos derived in vivo or in vitro.

OBJECTIVE: To develop an effective ICR mouse embryo culture medium. DESIGN: In vitro model study. SETTING: University-affiliated hospital. ANIMALS: Four-week-old, superovulated mice. INTERVENTION(S): In vivo- or in vitro-derived one-cell embryos were cultured in preimplantation-1 medium (P-1). MAIN OUTCOME MEASURE(S): Preimplantation development. RESULT(S): In vivo-derived embryos were cultured in BSA-containing P-1, to which one of the following substances was added: [1] no addition, [2] amino acids (aa), [3] aa+hemoglobin (hb), [4] aa+hb+cysteine (cys), [5] aa+hb and glucose (glu) added at the four-cell, or [6] aa+hb and glu+cys added at the four-cell stage. More (P<0.05) blastocysts developed after aa or aa+hb addition than after no addition, and glu addition to such medium further stimulated the formation (54%). In P-1 with aa+glu, the addition of 1 microg/mL hb was optimal. Additional improvement of blastocyst formation (78%) was achieved by ethylenediaminetetraacetic acid (EDTA), supplementation and bovine serum albumin replacement with polyvinyl alcohol (PVA) did not inhibit the development. P-1 supplemented with aa, hb, glu, EDTA, and PVA also supported the development of in vitro-derived embryos (70%). CONCLUSION(S): A modified P-1 medium was developed, and it supported the development of both in vivo- and in vitro-derived ICR mouse embryos.

Amino Acids↗

Outcome of thaw embryo transfer after cryopreservation of all embryos in patients at risk of ovarian hyperstimulation syndrome.

OBJECTIVE: To determine the incidence of ovarian hyperstimulation syndrome (OHSS) and subsequent pregnancy rates (PRs) if ET is delayed in patients at risk of OHSS by allocating all embryos to cryopreservation. DESIGN: Retrospective analysis of clinical and laboratory data from 724 consecutive stimulated cycles of IVF. SETTING: University hospital-based IVF program. PATIENTS: Consecutive patients undergoing IVF between September 1989 and December 1992. MAIN OUTCOME MEASURES: Fertilization rates, cryosurvival rates, subsequent PRs, and the occurrence of severe OHSS. RESULTS: Ten of the 564 patients (1.8%) who had ET in the stimulation cycle developed severe OHSS. Sixty-nine patients had all embryos frozen because of the risk of OHSS, of which one (1.4%) developed severe OHSS. The subsequent PR after thaw transfer was 25.2% per transfer, with a cumulative PR per patient after additional thaw transfers of 40.6%. CONCLUSIONS: Cryopreservation of all embryos and delayed ET in patients at risk of OHSS results in a low incidence of severe OHSS. Oocyte quality, fertilization rates, and cryosurvival of frozen embryos are equal to those for patients who have a normal stimulation profile. Subsequent thaw embryo replacements result in a satisfactory PR.

Adult↗

Abdominal pregnancy on the bladder wall following embryo transfer with cryopreserved-thawed embryos: a case report.

OBJECTIVE: To describe the occurrence and management of an abdominal pregnancy of the bladder after ET with cryopreserved-thawed embryos. DESIGN: Case report. SETTING: Infertility program in a tertiary care hospital. PATIENT(S): A woman with secondary infertility and a history of breast cancer. INTERVENTION(S): Transfer of cryopreserved-thawed embryos was performed, serial serum hormonal measurements were obtained, methotrexate was given IM, operative laparoscopy was performed. MAIN OUTCOME MEASURE(S): Documentation of abdominal pregnancy after transfer of cryopreserved-thawed embryos and its successful laparoscopic management. RESULT(S): Abdominal pregnancy occurred after ET of cryopreserved embryos in a patient with mild tubal disease. Diagnosis and management using laparoscopy were achieved. CONCLUSION(S): Abdominal pregnancy can occur using ET of cryopreserved-thawed embryos in a patient with mild tubal disease. If anatomically accessible, such a pregnancy can be managed successfully laparoscopically.

Adult↗

Replacement of one selected embryo is just as successful as two embryo transfer, without the risk of twin pregnancy.

The transition of in vitro fertilization from research to standard clinical practice has, to a great extent, been as a result of the use of controlled ovarian hyper stimulation. A disadvantage of the availability of multiple embryos has been the replacement of several embryos leading to an epidemic of multiple pregnancies. This retrospective review of 2606 fresh embryo transfers between 2001 and 2003, where either one or two selected embryos were replaced from an available cohort of at least four, shows that single embryo transfers have a similar pregnancy rate without the risk of multiple pregnancy.

Adult↗

Do patients need to remain in bed following embryo transfer? The Birmingham experience of 103 in-vitro fertilization cycles with no bed rest following embryo transfer.

Since the early days of human in-vitro fertilization and embryo transfer, rest in bed for hours immediately following the transfer has been advocated and widely practised. However, there is no scientific validation for this practice which is both time-consuming for the patient and increases space occupancy in the hospital or clinic. We report here on a study of 103 in-vitro fertilization cycles with no bed rest in hospital following the embryo transfer. The mean number of embryos transferred was 2.7 (range 1-3) and the clinical pregnancy rate per embryo transfer procedure was 40%. These results suggest that bed rest is not necessary following embryo transfer.

Adult↗

Avian genetic resource banking: can fish embryos yield any clues for bird embryos?

Cryopreservation of avian germplasm is becoming better understood and more commonly practiced. However, one area that would be of great benefit for genome resource banking is the preservation of avian embryos. Little is know about the cryobiology of avian embryos, and they have never been successfully cryopreserved. However, it is likely that they share many of the challenges of other yolk-filled multicompartmental embryos. For example, the fish embryo has 1) a large overall size, resulting in a low surface-to-volume ratio, which retards water and cryoprotectant efflux/influx; 2) large-sized cells, such as the yolk, which could increase the likelihood of membrane disruption by intracellular ice formation; 3) compartments, such as the blastoderm and yolk, with differing permeability properties; and 4) susceptibility to chilling injury. Both the avian and fish systems share many physical and anatomical properties, and it is predicted that some of the same permeability barriers would exist in both as well. Although the systems are similar, some of the goals, and thus the practices, to protect the genome may be quite different. One of these major goals in avian developmental biology is to produce chicken:chicken transgenic animals, especially those with germ line transmission. Producing efficient germ line transmissions and being able to cryopreserve these transmissions would be extremely beneficial to both basic and agricultural science. This could be accomplished through the cryopreservation of embryonic gonadal tissue followed by grafting into a host. The gonadal/tail-graft system would provide an advantage for cryopreservation because it is small (in comparison with the whole embryo), has fairly uniform tissue, and contains the essential primordial germ line cells capable of recreating the genetic line of interest. Moreover, because the chicken is such a robust model for most other avian species, the cryopreservation of the gonadal/tail-graft may potentially open up similar treatments for other commercially important species.

Animals↗

Embryo survival following non-surgical embryo recovery in superovulated dairy heifers.

Embryo survival following non-surgical embryo recovery was studied in 66 superovulated dairy heifers. Blood samples were collected at uterine flushing (6.5 days after a single AI), 72 h after flushing and then every 7 days until estrus or until pregnancy diagnosis by palpation per rectum. Luteolytic effect by uterine flushing was detected in three heifers 72 h following flushing (progesterone values < 1 ng/ml plasma). From the remaining animals 11 (17.5%) remained pregnant and 52 returned to estrus. In four heifers, embryo loss was registered 48 days after AI. Seven heifers developed gestation to term: five resulted in single and two in twin normal births. Progesterone concentrations in all samples from heifers in which embryo loss occurred were higher than in those with pregnancy to term. For non-pregnant heifers, the average interval between uterine flushing and the following spontaneous estrus was 16.6 +/- 1.3 days (+/- SEM). It was concluded that uterine flushing did not induce luteolysis in most heifers, and after uterine flushing, embryos remaining in the uterus were capable of developing through pregnancy to term.

Animals↗

Inhibition of chick embryo hepatic uroporphyrinogen decarboxylase by components of xenobiotic-treated chick embryo hepatocytes in culture. II.

A variety of xenobiotics, viz., 3,3',4,4'-tetrachlorobiphenyl (TCBP), sodium phenobarbital (PB), 3,5-diethoxycarbonyl-2, 4,6-trimethylpyridine (OX-DDC), and nifedipine, cause a decrease in uroporphyrinogen decarboxylase (UROG-D) activity, accompanied by uroporphyrin accumulation, in chick embryo hepatocytes in culture. In this study the activity of 17-day-old chick embryo hepatic UROG-D was determined by measuring the conversion of pentacarboxylporphyrinogen I to coproporphyrinogen I, and it was shown that a UROG-D inhibitor, previously reported to accumulate in TCBP-treated and PB-treated chick embryo hepatocytes in culture, also accumulates in OX-DDC-treated and nifedipine-treated chick embryo hepatocytes in culture. It was concluded that the accumulation of a UROG-D inhibitor provides an explanation for the UROG-D inhibition observed in this culture system with xenobiotics that cause uroporphyrin accumulation. Studies of the UROG-D inhibitory fraction isolated from the 10,000 x g, 40,000 x g, and 100,000 x g supernatant fractions of cultured chick embryo hepatocyte homogenate led to the conclusion that the UROG-D inhibitor is derived from a soluble component of the homogenate.

Animals↗

Brief cytochalasin-induced disruption of microfilaments during a critical interval in 1-cell C. elegans embryos alters the partitioning of developmental instructions to the 2-cell embryo.

We are investigating the involvement of the microfilament cytoskeleton in the development of early Caenorhabditis elegans embryos. We previously reported that several cytoplasmic movements in the zygote require that the microfilament cytoskeleton remain intact during a narrow time interval approximately three-quarters of the way through the first cell cycle. In this study, we analyze the developmental consequences of brief, cytochalasin D-induced microfilament disruption during the 1-cell stage. Our results indicate that during the first cell cycle microfilaments are important only during the critical time interval for the 2-cell embryo to undergo the correct pattern of subsequent divisions and to initiate the differentiation of at least 4 tissue types. Disruption of microfilaments during the critical interval results in aberrant division and P-granule segregation patterns, generating some embryos that we classify as 'reverse polarity', 'anterior duplication', and 'posterior duplication' embryos. These altered patterns suggest that microfilament disruption during the critical interval leads to the incorrect distribution of developmental instructions responsible for early pattern formation. The strict correlation between unequal division, unequal germ-granule partitioning, and the generation of daughter cells with different cell cycle periods observed in these embryos suggests that the three processes are coupled. We hypothesize that (1) an 'asymmetry determinant', normally located at the posterior end of the zygote, governs asymmetric cell division, germ-granule segregation, and the segregation of cell cycle timing elements during the first cell cycle, and (2) the integrity or placement of this asymmetry determinant is sensitive to microfilament disruption during the critical time interval.

Actin Cytoskeleton↗

Prostaglandin production by horse embryos and the effect of co-culture of embryos with endometrium from pregnant mares.

Embryos, endometrial biopsies, and uterine lavage fluid were collected from pregnant and non-pregnant mares 14 days after ovulation. Embryos were cultured for 20.5 h with and without endometrial tissue from pregnant mares, and endometrial tissue was cultured alone. Endometrial content of PGF tended to be higher (P = 0.06) in non-pregnant than in pregnant mares, but the amount of PGF released from tissue during culture was similar for pregnant and non-pregnant mares. Lavage fluid from non-pregnant mares also tended (P = 0.08) to contain higher concentrations of PGF. Coincubation of embryos with endometrium from pregnant mares significantly (P = 0.01) lowered concentrations of PGF in medium. Tissue concentrations and release of PGE-2 and 6-keto-PGF-1 alpha were similar in endometrial samples from pregnant and non-pregnant mares and prostaglandin production was unaffected by the presence of an embryo during incubation. Horse embryos released all three prostaglandins during a 20.5-h incubation.

6-Ketoprostaglandin F1 alpha↗

Embryo associated immunosuppressor factor(s) secreted by preembryo and serum estradiol levels are predictive of pregnancy outcome: effect of gonadotropin releasing hormone agonist (GnRHa) treatment of patients undergoing in vitro fertilization and embryo transfer (IVF-ET).

The aim of the present study was to determine whether (i) the secretion of embryo associated immunosuppressor factor (EASF) by preimplantation embryo and serum estradiol (E2) play a cooperative role in pregnancy outcome and (ii) whether this association was affected by gonadotropin releasing hormone agonist (GnRHa) pretreatment of IVF-patients for ovarian stimulation. EASF activity was measured using concanavalin A-induced lymphocyte proliferation assay in 251 IVF-culture media obtained from 59 patients undergoing IVF-ET. EASF activity in embryo growth media, cycle day 3 serum E2 level and peak E2 levels were correlated with pregnancy outcome. Results indicate that in GnRHa non-treated patients (i) the immunosuppressive activity of embryo growth media significantly (p less than 0.05) differed between the patients with varying concentration of day 3 serum E2, (ii) the presence of immunosuppressive activity in the embryo growth media was associated with success of pregnancy in patients with day 3 serum E2 level of 21-40 pg/ml (p less than 0.005) and in patients with peak E2 level in serum of less than = 1500 pg/ml (p less than 0.05). No association in immunosuppressive activity, serum E2 level and successful pregnancy was seen in GnRHa treated patients. This preliminary study suggests that the concentration of serum hormones and factors that are altered due to GnRHa pretreatment of IVF-patients may affect the cooperative role played by EASF, E2 and other factors in the success of pregnancy.

Blastocyst↗

The influence of egg sequence position on fertility, embryo viability, and embryo weight in broiler breeders.

A study was undertaken to determine whether fertility, early embryo viability, and the weight of the egg, shell, and embryo are influenced by the position of an egg in a laying sequence. One hundred and three Indian River broiler breeder hens were housed individually in cages at 21 wk of age. Beginning at 43 wk of age, each hen was inseminated once per week, with 50 microL of fresh undiluted pooled semen from Indian River broiler breeder males. Beginning at 45 wk of age, and for the following 45 days, all eggs laid were collected. Eggs were collected hourly for 8 h after "lights on" and a final daily collection was made 14 h after lights on. Each egg was classified as "first-of-sequence" or "subsequent". Eggs were weighed and placed in a forced-air incubator twice per week. After 168 h of incubation, the eggs were removed from the incubator, weighed and assessed for fertility and embryo viability status. The embryo was also weighed. Shell weight was recorded after 3 to 4 days of air drying. Egg weight was significantly greater in first-of-sequence compared with subsequent eggs. First-of-sequence eggs exhibited significantly smaller Day 7 embryos (on a percentage basis), lower shell weight (on a percentage basis), and significantly less egg weight loss during the 7 day incubation period (absolute and percentage basis) than did subsequent eggs on a sequence. Least squares (LS) means for fertility did not differ significantly between first (87.02%) and subsequent (89.56%) eggs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

[A study on the relationship between serum anticardiolipin antibody and embryo implantation in an in vitro fertilization and embryo transfer program].

OBJECTIVE: To study the role of serum anticardiolipin antibody in embryo implantation in an in-vitro fertilization and embryo transfer program. METHODS: 150 in vitro fertilization and embryo transfer (IVF-ET) treatment cycles from Nov. 1994 to May, 1996 were studied. Serum anticardiolipin antibody was measured by enzyme-linked immunosorbent assay (ELISA), and the clinical pregnancy outcome observed. RESULTS: Lower pregnancy rate 9.5% was found in 21 cycles of anticardiolipin antibody seropositive women compared with 26.3% in 129 cycles of anticardiolipin antibody seronegative women (P < 0.05). Patients with biochemical pregnancy and no pregnancy had seropositive anticardiolipin antibody in 20.0% and 16.2% compared with 5.6% in the patients with clinical pregnancy (P < 0.05). CONCLUSIONS: Failure of embryo implantation is associated with many factors. Our study demonstrated the serum anticardiolipin antibody in patients may play a part in embryo implantation and very early postimplantation loss.

Antibodies, Anticardiolipin↗

Transplantation of nuclei from 2- to 16-cell embryos into enucleated blastomeres of 2-cell mouse embryos.

Individual blastomeres of 2-, 4-, 8- and 16-cell embryos were fused with enucleated blastomeres of 2-cell embryos. The percentage of fused nuclear donor/recipient pairs was high (92 to 100%). Most of the reconstituted embryos cleaved twice within 24 h of in vitro culture and 83% to 95% developed to the morula or blastocyst stage. Increasing the number of blastomeres in reconstituted embryos by the construction of aggregates from blastomeres obtained 24 h after nuclear donor/recipient fusion was effective. All constructed aggregates reached the morula or blastocyst stage within 30 h of in vitro culture. The normal day-13 implantations were recorded after the transfer of reconstituted "half" embryos and aggregates when 8-cell blastomeres were used as nuclear donors.

Animals↗

Timing of the initiation of rRNA gene expression and nucleolar formation in cleavage embryos arrested by cytochalasin B and podophyllotoxin and in cytoplasm-extracted embryos of Xenopus laevis.

The timing mechanism of rRNA gene expression was studied in Xenopus laevis embryos whose cleavage was arrested by treatment with cytochalasin B or podophyllotoxin, and in fertilized eggs from which about 40% of the cytoplasm was extracted by micromanipulation. rRNA synthesis was activated in the cleavage-arrested embryos and also in cytoplasm-extracted embryos at the time when the control embryos commenced synthesizing rRNA at the late blastula stage. Formation of the nucleolar structure occurred at the normal time in the nuclei of cleavage embryos arrested by podophyllotoxin. Therefore, neither twelve continuous cell divisions nor the nuclear division which normally precedes the MBT (mid-blastula transition) was the factor determining the timing of rDNA expression. Moreover, lowering of the cytoplasmic:nuclear ratio to a certain threshold level was not a prerequisite for rDNA activation. Some regulatory mechanism dependent on the time elapsed from fertilization is likely to be the important factor permitting rDNA expression.

Animals↗