Judy Fleetham

Purified human early pregnancy factor from preimplantation embryo possesses immunosuppressive properties

This study was undertaken to determine whether early pregnancy factor secreted by preimplantation embryos has immunosuppressive properties. Human early pregnancy factor was purified from embryo growth media of in vitro fertilized ova with ion-exchange and gel filtration chromatography. During each step of purification the fractions were tested for (1) early pregnancy factor activity with the rosette inhibition assay, (2) immunosuppressive properties with a concanavalin A-stimulated lymphocyte proliferation assay, and (3) purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results indicate that (1) human early pregnancy factor has a basic molecular weight of 14 kd, (2) early pregnancy factor has immunosuppressive activity, (3) polymers of early pregnancy factor also appear to be present in the embryo growth media, and (4) immunosuppressive factors other than early pregnancy factor are also secreted by preimplantation human embryos. Early pregnancy factor and other factor(s) produced by the preimplantation embryo may play a role in suppressing maternal cellular immune responses, thereby preventing maternal rejection of the embryo.

A prospective randomized trial of conventional in vitro fertilization versus intracytoplasmic sperm injection in unexplained infertility

Purpose: To compare outcomes in patients with unexplained infertility undergoing conventional in vitro fertilization (IVF) versus intracytoplasmic sperm injection (ICSI). Methods: Sixty women with unexplained infertility in a Canadian tertiary-level clinic were randomized to IVF or ICSI. Subjects underwent downregulation with gonadotropin-releasing hormone agonist prior to initiation of recombinant human follicle-stimulating hormone. The primary outcome measure was fertilization rate. Secondary outcomes included implantation rate, embryo quality, clinical pregnancy rate, and live birth rate. Results: There was no statistically significant difference in fertilization rate (77.2% IVF vs. 82.4% ICSI), implantation rate (38.2% IVF vs. 44.4% ICSI), clinical pregnancy rate (50% in each group), or live birth rate (46.7% IVF vs. 50% ICSI). There were two cases of failed fertilization in the IVF group. There was no significant difference in embryo quality between groups. Conclusions: There were no differences in clinical outcomes associated with IVF versus ICSI in the treatment of unexplained infertility.

Growth of mouse embryos in bicarbonate media buffered by carbon dioxide, hepes, or phosphate.

The purpose of this study was to determine if mouse embryos could be grown successfully in a culture medium devoid of the carbon dioxide phase (CO2). Mouse embryos fertilized in vivo were collected and cultured in Hepes medium with and without bicarbonate (HCO3−) and a phosphate medium with and without HCO3−. In these experiments no CO2 gas phase was used. Further embryos were cultured in Whittinghams modified Tyrodes (T6) medium with a CO2 gas phase and served as controls. The degree of embryonic development was noted. Surviving blastocysts were transferred to the uteri of pseudopregnant mice and delivery at term was allowed to occur. There was no significant difference in the degree of embryonic development in those embryos cultured in T6 or Hepes medium (+HCO3−) or in the number of live offspring obtained when these blastocysts were placed within the mouse uterus. Although embryonic development apparently proceeded successfully in the phosphate (+HCO3−) medium, none of these blastocysts survived when transferred to mouse uteri. No embryonic growth occurred in either the Hepes or phosphate media which were devoid of HCO3−. It appears that a Hepes medium containing HCO3−, which uses no CO2 gas phase, is as effective as T6 medium, which uses a gas phase, in supporting in vitro mouse embryonic growth.

Protein synthetic patterns in immature and mature human oocytes.

A preliminary study of protein synthesis and amino acid transport in human oocytes was initiated. Qualitative patterns or protein synthesis were examined in individual oocytes cultured in medium containing radiolabeled methionine. The protein synthetic profile of immature oocytes, resolved by one-dimensional electrophoresis and fluorography, was observed to change markedly following germinal vesicle breakdown and oocyte maturation. No further differences in the one-dimensional protein synthetic patterns were observed in mature oocytes maintained in culture from 10 hours up to as long as 50 hours. The protein synthetic pattern of follicular cells was observed to be distinct from that of oocytes and was characterized by the predominant synthesis of a polypeptide with Mr = 44,000. Based on the specific activity of the methionine precursor, the absolute rate of synthesis was calculated to be about 50 pg protein/oocyte/hour. Total protein content was measured to be about 150 ng/egg. Competition of methionine uptake by leucine, efflux of radiolabeled methionine from preloaded oocytes into medium containing methionine and uptake of methionine in medium with low sodium ion concentration was observed. These findings are consistent with the presence of an L (leucine-preferring) system for neutral amino acid transport, similar to that in mouse and rabbit eggs. These studies provide basic data for further analysis of oocytes and perhaps preimplantation-stage embryos in the future.

Effects of progesterone on luteinizing hormone release and estradiol/progesterone ratio in the luteal phase of women superovulated for in vitro fertilization and embryo transfer

The present study extends the information on the effects of progesterone (P) on the luteinizing hormone (LH) release, and estradiol (E2)/P ratio in the luteal phase in women superovulated for in vitro fertilization and embryo transfer (IVF-ET). Two groups of 34 patients were induced for ovulation with clomiphene citrate and human menopausal gonadotropins. One group was given 25 mg P (Gesterol, Steris Laboratory Inc., Phoenix, AZ) at the time of, or 4 to 6 hours before human chorionic gonadotropin (hCG) administration and another group served as control (no Gesterol). Of the 34 patients in the Gesterol group, 10 had Gesterol 4 to 6 hours before the administration of hCG, 13 at the time of hCG, and 11 after the spontaneous LH surge. Administration of Gesterol 4 to 6 hours before hCG significantly increased the LH values (19.0 +/- 10.3) compared with those who had Gesterol at the time of hCG (6.8 +/- 2.8, P = 0.0006). A single dose of Gesterol (25 mg P) significantly reduced the E2/P ratio during the luteal phase (P = 0.0005). However, the outcome of IVF-ET was the same in the Gesterol and no-Gesterol groups. It is concluded that a significant increase in P triggers an LH surge and a single dose of Gesterol decreases E2/P ratio in the luteal phase of women after ovarian stimulation. The biochemical mechanisms are unclear.

Transient hyperprolactinemia has no effect on endocrine response and outcome in in vitro fertilization (IVF)

Transient rises in plasma prolactin levels can be observed during the late follicular phases of both natural and stimulated cycles. It has been suggested that such a phenomenon might adversely affect the success of in vitro fertilization. This prospective study was designed to assess the effect of transient rises in prolactin levels on the endocrine response to ovarian stimulation and the outcome of in vitro fertilization treatment. A total of 90 treatment cycles in 87 couples was studied. Prolactin was measured in the mid and late follicular phases of the cycles. During the study period, 24 pregnancies occurred. There were no differences in those cycles in which pregnancy did or did not occur in either mid or late follicular prolactin levels. Neither the initial level nor the percentage rise in prolactin during the stimulation had any effect on the peak estradiol level achieved, the numbers of follicles seen, the number of eggs retrieved, or the incidence or outcome of pregnancy. It was concluded that transient hyperprolactinemia is of no significance in ovarian stimulation for in vitro fertilization.

The effect of the day of initiation of ovarian stimulation in the day of luteinizing hormone surge and outcome of in vitro fertilization

It was hypothesized that the day of initiation of ovarian stimulation may influence the day of the luteinizing hormone (LH) surge onset and follicular development. Two groups of 52 patients were randomly selected to commence ovarian stimulation on either day 2 or day 4. The mean +/- standard deviation day of the LH surge was 11.0 +/- 0.9 for day 2 and 12.2 +/- 0.9 for day 4 (P less than 0.001), and the day of human chorionic gonadotropin (hCG) administration was 10.7 +/- 1.2 for day 2 and 11.4 +/- 0.9 for day 4 (P less than 0.02). The two groups also differed significantly in the mean number of days of human menopausal gonadotropin (hMG) administration (day 2, 7.4 +/- 2.7, versus day 4, 6.3 +/- 2.5), and the mean number of vials of hMG administered (day 2, 10.4 +/- 3.2, versus day 4, 8.1 +/- 2.9). However, the mean estradiol level on the day of the LH surge or hCG administration, the number of oocytes collected and fertilized, the number of embryos transferred, and the pregnancy rates were not significantly different. In conclusion, the day of the LH surge or hCG administration can be influenced by the day of initiation of ovarian stimulation, and the initiation of ovarian stimulation around day 4 of the menstrual cycle is clinically more efficient than initiation of follicular development early in the follicular phase.

Purification of water for in vitro fertilization and embryo transfer

The purpose of this study was to determine whether water obtained from the Milli-Q water purification system (Millipore, Mississauga, Canada) needed further purification for use in in vitro fertilization and embryo transfer. We describe a method for maintenance of the Milli-Q system. To assess water quality, alternate batches of culture media were prepared by using either Milli-Q water or Milli-Q water further treated by twice glass distillation. The percentage of mouse two-cell embryos that developed to blastocysts and the human in vitro fertilization-embryo transfer pregnancy rates were recorded for each batch. There were no significant differences in the parameters examined, indicating that further purification by twice glass distillation is not necessary if the Milli-Q system has been maintained as outlined.

Recovery of a preovulatory binucleate oocyte in a patient following induction of ovulation for in vitro fertilization.

SummaryA preovulatory oocyte with two germinal vesicles was recovered. This oocyte underwent germinal vesicle breakdown but did not form a polar body.

Production and characterization of monoclonal antibodies to embryo-associated immunosuppressor factor (EASF) produced by human pre-implantation embryo

Balb/c mice were immunized with pre-implantation embryo-associated immunosuppressor factor (EASF) (purified from embryo growth media of in vitro fertilized human ova). Hybridoma clones were produced by fusing their spleen cells with NS1 and P3X653 myeloma cell lines. The presence of specific anti-EASF monoclonal antibodies (mAb) in the hybridoma culture supernatants were tested by enzyme-linked immunosorbent assay. A total of 15 hybridoma clones were selected, and their products were purified and characterized. Each mAb bound specifically to its antigen in a dose-dependent manner. The affinity-purified EASF from embryo growth media demonstrated immunosuppressive activity on Concanavalin A-induced lymphocytes and the presence of 14 kDa, 24 kDa and 37 kDa factors. No such activity or similar molecules were identified when control growth media were analyzed. This clearly demonstrates that these mAb are indeed EASF-specific and are able to recognize biologically active immunosuppressive components in embryo growth media. These mAbs are presently being tested for the development of EASF-specific assay system.