Steven R. Bayer

Characterization of insulin-like growth factor binding to human granulosa cells obtained during in vitro fertilization.

Insulin and IGF-I affect in vitro ovarian stromal and follicular cell function in several species. We previously characterized insulin receptors on human granulosa cells obtained from in vitro fertilization procedures but were unable to demonstrate specific binding of IGF-I. Following modification of the assay conditions, we now report specific, high affinity IGF-1 binding sites on human granulosa cells. Substitution of equimolar concentrations of sucrose for sodium chloride in the buffer solution increased binding of IGF but not insulin in equilibrium assays. Maximal specific IGF-I binding was 2.69 +/- 0.30%/10(5) cells (SEM, n = 9) with half-maximal inhibition of binding at 2 ng/ml IGF-I. Unlabeled insulin recognized the type I IGF receptor with low affinity. An IGF-I receptor monoclonal antibody (alpha IR-3) inhibited 125I-IGF-I but not 125I-insulin binding. Affinity crosslinking followed by SDS/PAGE under reducing conditions revealed IGF-I binding at a molecular weight compatible with the alpha subunit of the type I IGF receptor and with a pattern of inhibition by various ligands that paralleled the equilibrium binding assays. IGF-I receptors are present on freshly isolated human ovarian granulosa cells obtained following pharmacologic stimulation with gonadotrophin according to the protocols of in vitro fertilization. The biologic function of these receptors currently is being investigated.

The effect of endometriomas on in vitro fertilization outcome

To determine the effect of ovarian endometriomas on in vitro fertilization (IVF) outcome, two groups of patients were studied. Group I consisted of seven patients with ovarian endometriomas and severe pelvic adhesions treated for a total of 12 cycles. Group II patients consisted of eight patients with hydrosalpinges and comparable pelvic adhesions treated for a total of 27 cycles. There were no differences in the number of days required for stimulation or in the serum estradiol levels attained between the two groups. Group I patients were noted to have significantly fewer preovulatory follicles (1.42 vs 3.33,P<0.005), cycles with fertilization (28 vs 84%,P<0.005), and embryos transferred (0.78 vs 2.56,P=0.01) than Group II patients. Three pregnancies occurred in Group II, while there were no conceptions among Group I patients. This study suggests that the presence of an ovarian endometrioma(s) has an adverse effect on IVF outcome and suggests that patients with ovarian endometriomas should have them removed prior to undergoing IVF.

Spectrophotometric absorbance of follicular fluid: a predictor of oocyte fertilizing capability

Follicular fluid volume, scoring of the oocyte-cumulus-corona-complex (OCCC), and spectrophotometric absorbance of the follicular fluid were separately compared between a group of fertilized (n = 53) and unfertilized oocytes (n = 35). Scoring of the OCCC and follicular fluid volumes was not found to be statistically different between the two groups. Spectrophotometric analysis of the follicular fluid in the visible spectrum demonstrated two peaks of maximum absorbance at 415 nm and 455 nm. The group of oocytes that fertilized was associated with follicular fluids that had significantly higher absorbances at 415 nm and 455 nm. In conclusion, follicular fluid volume and scoring of the OCCC were poor predictors of fertilizing capability; however, spectrophotometric absorbance, particularly at 455 nm, was positively correlated with oocyte fertilization.

Rapid identification of deoxyribonucleic acid sequence differences in cytochrome P-450 21-hydroxylase (CYP21) genes with denaturing gradient gel blots

Denaturing gradient gel electrophoresis was used to identify single-base differences in the cytochrome P-450 21-hydroxylase (CYP21) genes of 132 unrelated control individuals and family members of three unrelated patients with 21-hydroxylase deficiency. The salt-wasting variety was caused by gene deletion and gene conversion/deletion mutations in affected members of two families studied. The simple virilizing form, present in the third family, was caused by an apparent point mutation not detectable by routine Southern blots. We have detected many restriction fragment melting polymorphisms in the CYP21 genes of the members of both salt-wasting families and normal individuals with denaturing gradient gel electrophoresis. We also identified a restriction fragment melting polymorphism specific for the simple virilizing patient in the third family. The data demonstrate that the CYP21 genes are highly polymorphic and that denaturing gradient gel electrophoresis is useful for genomic deoxyribonucleic acid analysis of patients with 21-hydroxylase deficiency.

IVF and embryo development subsequent to ovarian torsion occurring during the resumption of meiosis

This report describes an unusual case of ovarian torsion during an IVF cycle prior to vaginal oocyte retrieval and the subsequent embryo development. A 27-year-old, whose husband carries a balanced translocation, presented on stimulation day 11 (day after human chorionic gonadotrophin administration) with signs of right ovarian torsion. Transvaginal ultrasound identified decreased right ovarian venous flow but preservation of right ovarian arterial flow. She underwent emergency laparoscopic right ovarian detorsion followed by vaginal oocyte retrieval on postoperative day 1. Ten oocytes were retrieved from the right detorted ovary, 4/10 (40%) were fertilized and 3/4 (75%) became blastocysts. Fifteen oocytes were retrieved from the left ovary, 14/15 (93%) were fertilized and 9/14 (64%) became blastocysts. All 18 embryos biopsied for preimplantation genetic diagnosis carried unbalanced translocations and none were transferred. The markedly reduced fertilization rate of the oocytes from the previously torted ovary is similar to the rate described in a prior report and likely related to decreased but maintained ovarian arterial flow. This report is unique because not only was the patients ovarian torsion surgically corrected prior to oocyte retrieval but also the embryos originating from the previously torted ovary had excellent development with 75% progressing to the blastocyst stage.

Rubella susceptibility of an infertile population

The recent trend reported by the CDC reaffirms that rubella continues to be a health care issue and should be a concern particularly for those who care for women in the childbearing age group. Immunization of all susceptible women will not only offer them protection but reduce the pool of susceptible individuals that can transmit the disease.

Spectrophotometric analysis of follicular fluid related to oocyte fertilization, embryo cleavage, and follicular fluid protein and hormone content**Presented at the 6th World Congress on In Vitro Fertilization and Embryo Transfer, Jerusalem, Israel, April 3 to 7, 1989.

Spectrophotometric absorbance patterns of follicular fluids (FF) obtained from in vitro fertilization patients were analyzed and compared with oocyte fertilization and embryo cleavage rates. Each absorbance pattern was resolved into three components: delta OD360, delta OD415, and delta OD455. A positive correlation was found between fertilization outcome and delta OD455. The absorbances were not related to embryo cleavage rates. We confirmed the presence of bilirubin and beta-carotene in FF: these pigments absorb at 455 nm and are most likely serum-derived. To explain the association between delta OD455 and fertilization, we hypothesized that the FF delta OD455 is a marker of the degree of vascularization of the follicle that could be assessed by FF protein and hormone concentrations. However, no correlation was found between the delta OD455 and these FF parameters, suggesting an alternative explanation for this association.