Embryonic platelet-activating factor: an indicator of embryo viability.

BACKGROUND: A definitive need exists to identify a biomarker of embryonic viability. Platelet-activating factor (PAF) production by human embryos is related to pregnancy potential. METHODS: Conditioned embryo culture media were obtained following conventional IVF on day 3, with PAF levels and pregnancy outcomes correlated. RESULTS: Overall pregnancy rate was 68% (17/25) with a mean of 84.1 (+/- 8.5) pmol/l/embryo PAF level. PAF levels ranged from a 216.4 pmol/l/embryo (pregnant) to a 3.7 pmol/l/embryo (not pregnant). There was a significant difference (P < 0.05) in PAF content between pregnant (92.1 +/- 9.5 pmol/l/embryo) and non-pregnant groups (52.5 +/- 16.6 pmol/l/embryo). Patients were categorized into three groups based upon PAF levels: low (< or= 5 pmol/l/embryo); medium (51-100 pmol/l/embryo) and high (>100 pmol/l/embryo). The low (60%) group had a significantly (P < 0.05) lower pregnancy rate than either the medium (85%) or high (89%) groups. A receiver-operator characteristic curve predicted a cut-off limit of 45 pmol/l/embryo for PAF content in human embryo conditioned culture media. CONCLUSIONS: The data demonstrate a correlation between PAF levels in human embryo conditioned culture media and pregnancy outcome. Additionally, as embryonic PAF levels increase so does the corresponding pregnancy rate. Therefore, PAF may be used as an indicator of embryo viability and for predicting pregnancy outcome.

Cryopreservation of immature and mature human oocytes.

Cryopreservation of oocytes facilitates the long-term storage of oocytes for patients in danger of losing ovarian function. It also alleviates many of the ethical concerns associated with embryo cryopreservation. Problems associated with metaphase II oocyte cryopreservation include zona pellucida hardening and spindle damage. The cryopreservation of germinal vesicle-stage oocytes has been undertaken as a means of circumventing the problem of spindle damage in mature oocytes. One of the main disadvantages of immature oocyte cryopreservation is the fact that in vitro maturation is required post-thaw. The majority of live births from oocyte cryopreservation have involved the use of 1,2-propanediol and slow freezing protocols. Various methods have been used in an attempt to improve survival rates. These include vitrification and use of novel cryopreservatives. Future areas of concentration should include in vitro maturation, vitrification, and alternate cryopreservatives.

Simultaneous detection of chromosomes X, Y, 13, 18, and 21 by fluorescence in situ hybridization in blastomeres obtained from preimplantation embryos.

PURPOSE: Simultaneous fluorescence in situ hybridization (FISH) was used in a preimplantation genetic diagnosis program to determine which embryos were normal for chromosomes X, Y, 13, 18, and 21. METHODS: Single blastomeres were disrupted and attached to glass slides using acetic acid and ethanol. Using a ratio mixture of chromosome enumeration DNA probes in combination with locus-specific identifier DNA probes, FISH was performed for the identification of chromosomes X, Y, 13, 18, and 21. RESULTS: Fourteen couples enrolled in IVF produced 134 embryos for biopsy. Blastomeres subjected to five-color FISH revealed that 22% of embryos were normal for chromosomes X, Y, 13, 18, and 21. In addition, 52% were abnormal and no results could be detected for 25%. Twelve couples underwent embryo transfer, two couples did not receive embryos due to lack of any normal embryos, and three couples became pregnant. CONCLUSIONS: The simultaneous detection of five-color FISH is a feasible method to detect aneuploidy in preimplantation embryos from women of advanced maternal age.

Reinsemination by intracytoplasmic sperm injection of 1-day-old oocytes after complete conventional fertilization failure.

OBJECTIVE: To analyze the efficacy of intracytoplasmic sperm injection (ICSI) for "rescue" of failed conventional insemination cycles. DESIGN: Retrospective clinical study. SETTING: Private infertility clinic. PATIENT(S): Fifty-four couples with non-male factor infertility undergoing routine IVF within our clinic. INTERVENTION(S): Twenty- to 24-hour-old unfertilized mature oocytes from conventional IVF cycles in which fertilization failure was complete were reinseminated by ICSI to attempt late fertilization and rescue of the otherwise failed IVF cycle. MAIN OUTCOME MEASURE(S): Late fertilization, pregnancy, and embryonic implantation. RESULT(S): Intracytoplasmic sperm injection reinsemination of 489 unfertilized mature oocytes caused degeneration in 50 (10.2%) oocytes, gave rise to normal fertilization in 215 (44.0%) of the injected oocytes, and allowed ET in 48 of the 54 cycles in which initial complete fertilization failure had occurred. One hundred sixty-four (76.3%) of 215 late-fertilized oocytes either were used for fresh transfer or were frozen-stored. Eight viable pregnancies resulted, yielding a 14.8% pregnancy rate per initiated cycle. CONCLUSION(S): Barring potential concerns regarding the chromosomal normality of embryos arising from reinsemination, our results suggest that ICSI is a relatively successful means of rescuing conventional IVF cycles in which fertilization fails completely.

Effect of coculture on subsequent survival and implantation of cryopreserved human embryos.

PURPOSE: This retrospective analysis was designed to assess the performance of human embryos following cryopreservation based on whether they were originally developed in standard culture medium (65 cycles, 223 embryos) or cocultured on partial monolayers of bovine oviductal epithelial cells (63 cycles, 198 embryos). Embryo cryosurvival and implantation were compared between the study group and the contemporaneously match controls. RESULTS: During a 2-year period when no factors of the cryopreservation program were altered, 63 transfers of 159 surviving thawed control cleavage-stage embryos (71.3% survival) that were 54% intact gave rise to 11 viable pregnancies (17.5%/ET), to yield an implantation rate of 6.9% per embryo. Sixty-three transfers of 147 thawed cocultured embryos (74.2% survival) that were 61% intact gave an implantation rate of 13.6% per embryo that was significantly higher than the control group (P < 0.05). CONCLUSION: Coculture of embryos prior to cryopreservation does not appear to improve cryosurvival; however, it does improve implantation postthaw compared with embryos following standard culture prior to cryopreservation.

Repeated sperm injection under the zona following initial fertilization failure.

When fertilization fails following micromanipulative under-zona insemination, it is possible to repeat the procedure adding more spermatozoa to achieve fertilization, embryonic development and pregnancy. We report on 18 human in-vitro fertilization cycles where this approach was used. In nine cycles only late-fertilized embryos were available for transfer, and these gave rise to two viable pregnancies (22.2% per transfer). In six cycles, where a mixture of late- and timely fertilized embryos were available for transfer, two viable pregnancies arose (33.3% per transfer). In three cycles no fertilization was achieved even after reinsemination by repeated under-zona insemination.

Electroejaculation in combination with in vitro fertilization and gamete micromanipulation for treatment of anejaculatory male infertility.

OBJECTIVE: Failure to ejaculate may be overcome by use of electroejaculation. However, such semen samples are often unsuitable for therapies like intrauterine insemination. The combination of electroejaculation with in vitro fertilization, including gamete micromanipulation, should improve chances of fertilization and pregnancy in such cases. STUDY DESIGN: Within a private infertility clinic electroejaculation in combination with intrauterine insemination was carried out in 18 cycles (10 couples). Four couples went on to receive therapy by electroejaculation plus in vitro fertilization, along with six other couples (15 cycles total) with semen too poor for intrauterine insemination. RESULTS: One term pregnancy arose in the electroejaculation-intrauterine insemination group, and one term pregnancy plus one continuing pregnancy arose from two couples (three cycles) who underwent in vitro fertilization with conventional insemination after electroejaculation. Six couples (nine cycles) had embryos arising only from gamete micromanipulation transferred, and this yielded two term pregnancies, one spontaneous abortion, and a biochemical pregnancy. Two couples (three cycles) failed to achieve fertilization even with micromanipulation; however, donor-inseminated eggs gave rise to two term pregnancies and one continuing pregnancy in these patients. CONCLUSIONS: This report confirms the feasibility of in vitro fertilization in conjunction with electroejaculation and extends the therapy to incorporate gamete micromanipulation.

Cryopreserved zygotes and embryos and endocrinologic factors in the replacement cycle.

Several parameters relating to features of replacement cycles in 88 patients were found to influence implantation of cryopreserved zygotes and early cleaved embryos. Cryopreserved embryos were replaced in 47 patients with a natural cycle, resulting in 12 (25%) clinical pregnancies. Patients who had anovulation or irregular cycles received either 50 mg clomiphene citrate on days 5 to 9 or 100 mg on days 2 to 6; the incidence of clinical pregnancy was 7 of 23 (30%) and 2 of 18 (11%), respectively. Neither a rise in follicular phase estradiol (E2) nor absolute levels of E2 predicted implantation. The length of the follicular phase during the replacement cycle correlated well with previous menstrual cycles in 43 (54%) of the patients, and 16 (37%) of these patients became pregnant. The follicular phase was either longer or shorter than anticipated in 39 patients, and only 5 (13%) became pregnant: a detrimental effect was especially apparent when the follicular phase was shortened.

Cryopreservation of zygotes and early cleaved human embryos.

Zygotes and 2- to 5-cell human embryos were frozen in 1,2-propanediol and sucrose; results of the first 50 cycles (45 patients) are presented. A total of 41 zygotes (17 attempts at thawing) were thawed, resulting in six singleton clinical pregnancies (15% per embryo; 35% per cycle), of which three delivered, one aborted, and two are ongoing. Fifty-seven cleaved embryos were thawed in 33 other cycles, resulting in four singleton and one twin pregnancy (11% per embryo; 15% per cycle), of which four delivered and one is ongoing. Depending on the cell stage, 61% to 81% of embryos survived cryostorage, but 2-cell embryos did not implant. One fifth of cryoinjury was due to the formation of cracks in the zona pellucida. The incidence of implantation was not enhanced when more than one freeze/thawed embryo was replaced, most pregnancies being obtained from single embryo replacements. At least 8% more births are expected in addition to conventional in vitro fertilization methods when the current policy of replacing three fresh embryos and freezing the remainder using this technique is applied. This method will result in two to four times more pregnancies per spare embryo, compared with other cryopreservation methods using older embryos.

An appraisal of warfarin therapy during pregnancy.

A retrospective analysis of 40 patients with cardiac disease who received sodium warfarin (Coumadin) therapy during pregnancy has been performed. The fetal mortality was 12,5%, but no case of congenital epiphyseal stippling was detected. The incidence of maternal postpartum haemorrhage was high. Warfarin should whenever possible be withheld during the 1st trimester of pregnancy and replaced by heparin.

The use of synthetic luteinizing hormone-releasing hormone to induce ovulation.

The therapeutic use of synthetic luteinizing hormone-releasing hormone (LH-RH) to induce ovulation has been explored. Three dosage schemes have been compared: a single dose of LH-RH, multiple doses of LH-RH, and LH-RH used in combination with human menopausal gonadotrophins (HMG). The results of these three schemes are presented and compared; the last regimen has proved most successful, with a high pregnancy rate, a low incidence of multiple pregnancy and no evidence of ovarian hyperstimulation.