Ian L. Pike

Maternal blood platelet physiology and luteal-phase endocrinology as a means of monitoring pre- and postimplantation embryo viability following in vitro fertilization

The discovery that the fertilized mouse ovum triggers an increased demand for platelets and results in thrombocytopenia during the preimplantation phase of pregnancy provides a monitor for embryo survival and viability. This paper reports a study in which the platelet count was significantly reduced throughout the human preimplantation phase of pregnancy and returned to normal following embryo implantation. The human embryo was shown to produce a platelet activating factor in vitro which caused the reduction in platelet count after embryo transfer. This factor in the embryo culture medium could be measured using a bioassay which provided a means of assessing embryo viability prior to transfer. Some women showed no reduction in platelets after transfer. These embryos failed to produce a platelet activating factor in vitro and pregnancy was not established. Other women displayed a reduction in platelets following transfer but failed to become pregnant. All of these women had elevated luteal-phase plasma E2 levels compared to pregnant patients, which may have interfered with the implantation process. Our observations provide a possible rapid and simple means for monitoring the viability of human embryos cultured in vitro and the survival of embryos in utero.

SUPPLEMENTATION OF IN-VITRO FERTILISATION CULTURE MEDIUM WITH PLATELET ACTIVATING FACTOR

The supplementation of culture medium with platelet activating factor (PAF) on the subsequent implantation and pregnancy potential of pre-embryos produced by in-vitro fertilisation was studied. Pre-embryo culture medium was supplemented with 0 (control), 0.186, 0.93, or 1.49 mumol/1 PAF. Pre-embryos were transferred to PAF-containing medium 15-17 h after insemination (ie, just before syngamy) for 24 h and then transferred to the uterus. For 185 women receiving control pre-embryos, the pregnancy rate (positive beta human chorionic gonadotropin per oocyte retrieval) was 10.2%, while 166 women who received PAF treated pre-embryos (all concentrations combined) achieved a pregnancy rate of 17.5%. This difference was significant. The pregnancy rates per pre-embryo transferred were 6.1% and 9.4% for the control and PAF groups, respectively. The percentage of positive pregnancy tests that resulted in a viable pregnancy (presence of fetal heart at 8 weeks) was 78.9% in the controls and 75.9% in the PAF group. There was no difference in the average number of embryos transferred in either group. The increase in the pregnancy rate after a short exposure of pre-embryos to PAF in vitro suggests that PAF mediates pre-embryo development.

Fertilization rates using intracytoplasmic sperm injection are greater than subzonal insemination but are dependent on prior treatment of sperm

OBJECTIVE To determine a suitable method of sperm preparation for use in micromanipulation. To compare the fertilization rates of sibling oocytes inseminated by intracytoplasmic sperm injection (ICSI) and subzonal insemination (SUZI). DESIGN Two methods of gamete micromanipulation to overcome male factor infertility were compared. Within this study, three trials were conducted to determine the most suitable method of sperm preparation. This method then was used to complete the study. SETTING Procedures were performed in a teaching hospital research environment. PATIENTS Ninety-six patients were recruited for this study, undergoing 99 stimulation cycles. INTERVENTIONS Sibling oocytes were inseminated by subzonal sperm or intracytoplasmic injection. MAIN OUTCOME MEASURES Fertilization, zygote development, and pregnancy rates. RESULTS In the absence of manipulative pretreatment of sperm (trial 1), there was no difference in normal fertilization rates between ICSI and SUZI (19% and 25%, respectively). In the second trial there again was no sperm pretreatment for SUZI but, for ICSI, a polyvinylpyrrolidone (PVP) solution was used to reduce sperm velocity and the sperm tails were incised before injection. The fertilization rates were significantly different between ICSI (44%) and SUZI (17%). In the third trial, PVP was added to the sperm used for both types of insemination and the sperm tails also were incised for the ICSI insemination. Fertilization was again significantly different (16% for SUZI and 44% for ICSI). The trial 2 method of sperm preparation then was used to complete the study (trial 4) and confirmed the results of trial 2 (21% for SUZI and 42% for ICSI). The results suggest that ICSI can give improved fertilization compared with SUZI provided the sperm are treated before injection. No significant differences were found in the development rates of zygotes suitable for transfer or cryopreservation between the two micromanipulation methods.

Subzonal insertion of multiple sperm is a treatment for male factor infertility.

Objective To determine whether subzonal insemination of multiple sperm is an effective treatment for male factor infertility. Design Subzonal insemination by micromanipulation of sperm and oocytes for male factor patients. Patients Two hundred eight patients were selected on previous fertilization history and/or semen parameters. They could be assigned to one of three groups: group A had previous IVF failure, group B had a low fertilization rate with previous IVF, and group C had insufficient sperm for IVF. Interventions Oocytes were inseminated by subzonal insertion of multiple sperm. Main Outcome Measures Fertilization and pregnancy rates. Results There have been 208 patients treated through 332 treatment cycles. Of 2,789 oocytes inseminated, 784 oocytes were fertilized (28%) and 516 of these (66%) were monospermic. This represents normal fertilization in 207cycles (62% of total patient cycles). These 207cycles produced an average of 2.6±1.8 embryos. The resultant 487 embryos were either transferred or cryopreserved for future transfer. A total of 42 pregnancies (21% per ET) resulted from 200 ET procedures with 397 embryos. Miscarriages or elective terminations have occurred (11/42 pregnancies, 26% to date) and 21 deliveries have resulted in 26 babies. There are nine ongoing pregnancies. Three of the pregnancies have been from cryopreserved embryos. Group B patients had the highest fertilization rate (23%) but the lowest pregnancy rate (7%). No other significant relationships between patient group and outcome were found. Conclusions Subzonal insemination of multiple sperm is an effective treatment for some forms of male factor infertility.

Subzonal insertion of multiple sperm is a treatment for male factor infertility

OBJECTIVE To determine whether subzonal insemination of multiple sperm is an effective treatment for male factor infertility. DESIGN Subzonal insemination by micromanipulation of sperm and oocytes for male factor patients. PATIENTS Two hundred eight patients were selected on previous fertilization history and/or semen parameters. They could be assigned to one of three groups: group A had previous IVF failure, group B had a low fertilization rate with previous IVF, and group C had insufficient sperm for IVF. INTERVENTIONS Oocytes were inseminated by subzonal insertion of multiple sperm. MAIN OUTCOME MEASURES Fertilization and pregnancy rates. RESULTS There have been 208 patients treated through 332 treatment cycles. Of 2,789 oocytes inseminated, 784 oocytes were fertilized (28%) and 516 of these (66%) were monospermic. This represents normal fertilization in 207 cycles (62% of total patient cycles). These 207 cycles produced an average of 2.6 +/- 1.8 embryos. The resultant 487 embryos were either transferred or cryopreserved for future transfer. A total of 42 pregnancies (21% per ET) resulted from 200 ET procedures with 397 embryos. Miscarriages or elective terminations have occurred (11/42 pregnancies, 26% to date) and 21 deliveries have resulted in 26 babies. There are nine ongoing pregnancies. Three of the pregnancies have been from cryopreserved embryos. Group B patients had the highest fertilization rate (23%) but the lowest pregnancy rate (7%). No other significant relationships between patient group and outcome were found. CONCLUSIONS Subzonal insemination of multiple sperm is an effective treatment for some forms of male factor infertility.

Supplementation of in‐vitro fertilisation culture medium with platelet activating factor

abnormally elevated 5 and 20 months before the clinical manifestations of recurrence became evident. The maximal concentrations approached 3000 U per liter. The serum inhibin level remained undetectable in one patient who was disease-free for 11 years. Serum inhibin concentrations were also elevated in three women with amenorrhea and infertility that resulted from small granulosa-cell tumors. After the removal of the tumors, the serum inhibin levels in these women became normal, and fertility returned. There was a significant negative correlation between the serum concentrations of inhibin and follicle-stimulating hormone, in a manner consistent with the autonomous production of inhibin by granulosa-cell tumors. We conclude that granulosa-cell tumors produce inhibin. Since serum inhibin levels reflect the size of the tumor, measurements of inhibin can be used as a marker for primary as well as recurrent disease.

Use of a bioassay for embryo-derived platelet-activating factor as a means of assessing quality and pregnancy potential of human embryos**Supported by a grant from the Australian Research Grants Scheme (ARGS).

The production of an embryo-derived platelet-activating factor (PAF) was recently shown to have a correlation with embryo quality and viability. The detection of this factor was used as a means of examining the effect of various aspects of the in vitro fertilization and embryo transfer procedure on human preimplantation embryo quality. Embryos that resulted in pregnancy produced significantly higher levels of embryo-derived PAF in vitro than embryos that failed to result in pregnancy. Of a further 85 embryos, 43% had a level of embryo-derived PAF that fell in the same range as the embryos that resulted in pregnancy. The production of embryo-derived PAF was related to the type of treatment used to induce follicular development (with clomiphene citrate and human menopausal gonadotropin commencing on day 5 giving best results); the size and estradiol production of the follicles producing the embryo; the age of the embryo culture medium; and the morphology and cell number of the embryos.

Failure of implantation in human in vitro fertilization and embryo transfer patients: the effects of altered progesterone/estrogen ratios in humans and mice**Supported by grant D18215709 from the Australian Research Grants Scheme.

Daily blood samples were taken for progesterone (P) and estradiol (E2) measurements from women who showed a platelet response consistent with the presence of viable embryos after in vitro fertilization and embryo transfer procedures. A comparison of steroid levels between those women who became pregnant and those who did not revealed the following: at and after the time of transfer, women who failed to become pregnant had significantly higher E2 levels and a lower ratio of P/E2 than women who became pregnant. The P/E2 ratio was a better predictor of implantation failure than was the absolute level of either hormone. Experiments were done in mice to test the hypothesis that P could protect implantation of the embryo against the inhibitory effects of high E2. In mice, implantation was inhibited by relatively high levels of E2. This effect was overcome by concomitant administration of P. There was a significant dose-response-related interaction of P with the E2