Previously reported better fertilization rate after intracytoplasmic single sperm injection (ICSI) than after subzonal insemination of several spermatozoa was confirmed in a controlled comparison of the two procedures in 11 patients. Intracytoplasmic sperm injection was carried out in 150 consecutive treatment cycles of 150 infertile couples, who had failed to have fertilized oocytes after standard in-vitro fertilization (IVF) procedures or who were not accepted for IVF because not enough motile spermatozoa were present in the ejaculate. A single spermatozoon was injected into the ooplasm of 1409 metaphase II oocytes. Only 117 oocytes (8.3%) were damaged by the procedure and 830 oocytes (64.2% of the successfully injected oocytes) had two distinct pronuclei the morning after the injection procedure. The fertilization rate was not influenced by semen characteristics. After 24 h of further in-vitro culture, 71.2% of these oocytes developed into embryos, which were transferred or cryopreserved. Only 15 patients did not have embryos replaced. Three-quarters of the transfers were triple-embryo transfers. High pregnancy rates were noticed since 67 pregnancies were achieved, of which 53 were clinical, i.e. a total and clinical pregnancy rate of 44.7% and 35.3% per started cycle and 49.6% and 39.2% per embryo transfer. A total of 237 supernumerary embryos were cryopreserved in 71 treatment cycles.
The present investigation studied the influence of the blastocyst's state of activity on the "window" of implantation in the receptive uterus in the mouse. The receptive state of the uterus is defined as the limited time when the uterine milieu is favorable to blastocyst acceptance and implantation. In the mouse, implantation occurs on day 4 (day 1 = vaginal plug). Ovariectomy in the morning of day 4 prior to preimplantation estrogen secretion results in blastocyst dormancy and delayed implantation. These conditions are maintained by continued progesterone (P ) treatment but can be terminated with an injection of estrogen leading to blastocyst activation and subsequent implantation. Blastocyst transfers into intact pseudopregnant mice demonstrated that the window of implantation on day 4 remains open at least through 1800 h for normal day 4 blastocysts but only up to 1400 h for dormant blastocysts. These results suggested that the blastocyst's state of activity influenced the normally operative window of implantation in the receptive uterus. This finding was further confirmed by inducing conditions of delayed implantation in pregnant donors and pseudopregnant recipients. They were ovariectomized on the morning of day 4 and maintained with daily injections of P from days 5 to 7. On day 7, dormant blastocysts from P -treated delayed donors were transferred into the uteri of P -treated delayed pseudopregnant recipients at 1, 2, 4, or 8 h after an injection of 17 β-estradiol (E ). Dormant blastocysts transferred into delayed recipients at 1 h after E treatment resulted in implantation in most of the animals as compared to complete failure of blastocysts to implant after transfer to P -treated delayed recipients at 4 or 8 h after E treatment. However, implantation did occur in P -treated delayed recipients at these later hours of E treatment when the P -treated delayed donors also received E prior to blastocyst transfer. Furthermore, the majority of day 4 normal blastocysts implanted when transferred into P -treated delayed recipients even at 16 h after E treatment. Interestingly, day 7 dormant blastocysts cultured for 8 or 24 h for in vitro activation failed to implant after transfer to P -treated delayed pseudopregnant recipients at 4 or 8 h after E treatment, although they did implant after transfer at 1 h after E treatment. As expected, normal day 4 blastocysts failed to implant after transfer to P -treated delayed pseudopregnant recipients. Thus, these results establish that the blastocyst's state of activity alters the timing of implantation (window) in the receptive uterus. Thus, the window for successful implantation could be defined as a limited time span when the activated stage of the blastocyst is superimposed on the receptive state of the uterus. This window remains open for a shorter period for dormant blastocysts than for normal or dormant blastocysts after E activation. Furthermore, dormant blastocysts, which apparently achieved metabolic activation in vitro, failed to attain the same status as blastocysts activated in utero by E for implantation into the receptive uterus. A key finding of this investigation is that E induces very rapidly, but transiently (1 h), a factor(s) in the P -primed uterus that activates the dormant blastocysts for implantation in the receptive uterus.
Several newly generated mouse embryonic stem (ES) cell lines were tested for their ability to produce completely ES cell-derived mice at early passage numbers by ES cell tetraploid embryo aggregation. One line, designated R1, produced live offspring which were completely ES cell-derived as judged by isoenzyme analysis and coat color. These cell culture-derived animals were normal, viable, and fertile. However, prolonged in vitro culture negatively affected this initial totipotency of R1, and after passage 14, ES cell-derived newborns died at birth. However, one of the five subclones (R1-S3) derived from single cells at passage 12 retained the original totipotency and gave rise to viable, completely ES cell-derived animals. The total in vitro culture time of the sublines at the time of testing was equivalent to passage 24 of the original line. Fully potent early passage R1 cells and the R1-S3 subclone should be very useful not only for ES cell-based genetic manipulations but also in defining optimal in vitro culture conditions for retaining the initial totipotency of ES cells.
Objective: To determine if an increase in plasma P occurring before hCG administration might impair the outcome of IVF-ET. Design: Five hundred eighty-five IVF-ET cycles were prospectively studied for the occurrence of plasma P elevation before hCG administration. Setting: Tertiary institution, IVF-ET program, Hopital A. Beclere. Patients: Participating patients included IVF-ET candidates 23 to 42 years of age only, excluding the couples in whom a male factor was a primary or an accessory cause of infertility. Main Outcome Measures: To clarify the practical consequences on IVF-ET outcome of pre-hCG increases in plasma P, we studied 585 consecutive IVF-ET cycles. These were divided into two groups according to plasma P levels observed on the day of hCG administration; plasma P of 0.9 ng/mL (2.9 nmol/L) was taken as an arbitrary cutoff value. Group A included 485 IVF cycles in which plasma P was less-than-or-equal-to 0.9 ng/mL (2.9 nmol/L); group B included the remaining 100 cycles in which plasma P was >0.9 ng/mL (2.9 nmol/L). Results: The number of mature oocytes retrieved, the oocyte cleavage rate, and the number of embryos obtained were similar in groups A and B. In contrast to this apparent similarity in oocyte quality, a decrease in pregnancy rate (PR) and a trend for a decrease in embryo implantation rate were observed in group B in comparison with group A. Conclusions: The similar fertilization and cleavage rates obtained in groups A and B suggest that pre-hCG elevation in plasma P does not lead to decreased oocyte quality. Yet the lower PR observed when plasma P rises prematurely suggests that the prolonged but discrete elevation in plasma P occurring in these cases might alter endometrium receptivity to embryo implantation.
Objective: To investigate the relationship between the embryo number and morphology in conception cycles and the incidence of multiple pregnancies. Design: The study is based on information received from a computerized data base. Setting: In Vitro Fertilization Unit, Sapir Medical Center, Kfar Saba, Israel. Patients: A total of 117 consecutive pregnancies resulted from replacement of fresh embryos in our IVF-ET program. Main Outcome Measures: The impact of embryo quality, as assessed by morphological parameters, on the multiple pregnancy rate (PR). Results: Implantation rates positively correlated with the number and the quality of transferred embryos. However, no multiple pregnancies occurred when only two embryos were replaced. There were no multiple pregnancies when only embryos of low quality (grades 1 and 2) were transferred. Furthermore, there was no correlation between the number of replaced embryos of poor quality and the rate of implantation. The multiple PR increased from 10% when a mixture of high and low quality embryos were transferred to 30.76% when only embryos of highest quality were transferred. Conclusion: The implantation rate of transferred embryos is directly correlated with the morphological scoring. The results of the study suggest that the number of embryos transferred should be balanced against their morphological quality to reduce the rate of multiple pregnancies.
The antiphospholipid syndrome is characterized by thrombocytopenia, thrombosis, and recurrent fetal loss in association with anti-cardiolipin antibodies (ACAs) or lupus anti-coagulants. However, the causal role of these antibodies in the disease and the mechanisms by which the ACA may induce the syndrome are not clear. Recently, we have established an experimental mouse antiphospholipid syndrome induced by the mouse IgM monoclonal ACA designated 2C4C2. In the present study, we focused on the effects of immunization with the monoclonal ACA 2C4C2 on the outcome of pregnancies in BALB/c female mice. Four weeks after active immunization with the monoclonal ACA, a severe gestational failure with low pregnancy rates, low number of fetuses, and a high rate of resorptions was observed. Moreover, embryos obtained from the ACA-immunized females on day 3.5 of pregnancy were severely impaired, demonstrating developmental delay and abnormal morphology. These abnormal embryos failed also to develop in an in vitro implantation model. Furthermore, specific binding of the 2C4C2 ACA to the trophectoderm cell lineage of in vitro implanting normal embryos was observed. Thus, our studies demonstrate that the severe ACA-induced gestational failure results from an impairment of implantation and suggest that the ACA may react directly with the preimplantation embryos.
Several studies have provided evidence that epidermal growth factor (EGF) or transforming growth factor α (TGFα) mediate some of the physiological effects of estrogen. The object of the present study was to determine whether exogenous EGF could initiate embryo implantation in the rat, a response known to depend upon the action of estrogen in a progesterone-primed uterus. Immunocytochemical examination showed the presence of immunoreactive EGF, TGFα and the EGF receptor in luminal glandular epithelium of uteri on days 4,5 and 6 of pregnancy. EGF receptor was also present in the implanting embryo and in decidual cells of the uterine stroma. Attempts to initiate implantation by intravenous injection of murine EGF into ovariectomized or hypophysectomized delayed-implanting rats maintained with progesterone were unsuccessful. Implantation sites were found, however, in 24 of 33 (73 per cent) hypophysectomized progesterone-primed rats given 100 μg EGF immediately after intrauterine transfer of blastocysts from hypophysectomized delayed implanting animals. Although EGF is capable of initiating implantation in the delayed implanting rat model it remains to be determined whether this growth factor is responsible for the implantation inducing action of estrogen in vivo.
The delayed implantation model was used to study epidermal growth factor receptor(s) (EGF-R) in the mouse blastocyst. Delayed implantation and blastocyst dormancy were induced by ovariectomy on day 4 of pregnancy and were maintained by daily (days 5-7) injections of progesterone (P ). Blastocyst activation and implantation were initiated by coinjection of estradiol-17β (E ) with P on the 3rd day of delay. Immunostaining of EGF-R, autoradiographic detection of I-labeled EGF binding, and measurement of EGF-inducible subcellular protein tyrosine phosphorylation demonstrated the loss of EGF-R from blastocysts (dormant) recovered 24 h after ovariectomy or on the 3rd day of P -maintained delay. However, increased EGF-R levels were detected in blastocysts (activated) recovered 12 or 24 h after E injection. Blastocyst EGF-R mRNA levels were quantitated by reverse transcriptase (RT)-PCR and distribution of this mRNA was examined by in situ hybridization. To provide a homologous probe for these studies, a mouse EGF-R partial cDNA was cloned and used as the template for synthesis of antisense- and sense-strand EGF-R RNA. Quantitative RT-PCR demonstrated an 8- to 10-fold reduction in EGF-R mRNA copies per cell in dormant blastocysts. In contrast, an 8-fold increase in EGF-R mRNA copies per cell was detected in activated blastocysts 8 h after injection of E . In situ hybridization detected EGF-R mRNA in most cells of normal day 4 blastocysts but not in those of dormant blastocysts. These studies establish that expression of the EGF-R gene in mouse blastocysts is tightly regulated by maternal steroid hormonal status.