Progesterone synthesis by luteinized human granulosa cells in culture: the role of de novo sterol synthesis and lipoprotein-carried sterol.

Human granulosa cells cultured in the presence of estradiol, FSH, and LH secreted relatively little progesterone during the first 3-5 days of culture. The cells then underwent a morphological transformation associated with a 10-fold or more increase in progestin production. Cells not cultured with the tropic hormones usually produced progestin at a lower rate. Serum was required for the expression of maximal steroidogenic activity. Inhibitors of de novo sterol synthesis, ML-236B and 5-cholesten-3 beta,25-diol, did not affect progestin secretion by luteinized cells cultured in 20% serum, but these inhibitors did reduce [14C]acetate incorporation into sterols by 90%. ML-236B also did not affect hCG-stimulated progesterone secretion by the cells. However, when the cells were changed into lipoprotein-deficient medium, progestin production was markedly reduced within 24 h. The addition of low density lipoproteins (LDL) to the culture fluid restored progestin production, whereas high density lipoproteins tended to further inhibit steroid secretion. The effects of LDL on steroidogenesis were dose dependent and saturable. We conclude that luteinized human granulosa cells use LDL-carried sterol as the primary substrate for steroidogenesis.

Stimulation of progesterone synthesis in luteinized human granulosa cells by human chorionic gonadotropin and 8-bromo-adenosine 3',5'-monophosphate: the effect of low density lipoprotein.

Primary cultures of luteinized human granulosa cells reduced progestin secretion when taken from serum- and gonadotropin-containing medium into serum- and hormone-free medium. When added individually hCG, 8-bromo-cAMP and low density lipoprotein (LDL) stimulated progestin secretion by the cells after they spent 48 h in serum- and hormone-free medium. However, combinations of hCG or 8-bromo-cAMP and LDL were most effective in increasing steroidogenesis. The effects of hCG in enhancing steroidogenesis in the presence of LDL were first detectable after 3 h, but were most marked after 20 h of culture. hCG and 8-bromo-cAMP increased the conversion of [3H]cholesteryl linoleate, which had been incorporated into the core of LDL, into [3H]progesterone. hCG also stimulated cellular accumulation of LDL cholesterol, as assessed by incorporation of [1-14C]oleic acid into sterol esters or by measurement of total cellular cholesterol in the presence of amino-glutethimide to block steroidogenesis. In contrast to progesterone secretion, estradiol secretion was not affected by the addition of LDL in the absence or presence of 8-bromo-cAMP. We conclude that LDL cholesterol is required for maximal rates of progestin synthesis by human luteinized granulosa cells. When granulosa cells are stimulated by hCG, uptake of LDL cholesterol is promoted, and there is increased utilization of LDL cholesterol for steroid synthesis.

Human chorionic gonadotropin and 8-bromo-adenosine 3',5'-monophosphate stimulate [125I]low density lipoprotein uptake and metabolism by luteinized human granulosa cells in culture.

In nonsteroidogenic cells, cellular cholesterol requirements and sterol availability determine low density lipoprotein (LDL) receptor expression and LDL metabolism. We wished to learn if hCG and cAMP increase LDL metabolism by cultured luteinized human granulosa cells and whether this increase is dependent on enhanced metabolism of cellular cholesterol stores to steroid. Granulosa cells were cultured for 48 h in medium containing 20% human male serum and then for 48 h in serum- and hormone-free medium. The cells then received either fresh medium (no additions) or one of the following treatments: 500 mIU hCG/ml, 1.5 mM 8-bromo-cAMP, 100 micrograms aminoglutethimide (AG)/ml to inhibit cholesterol metabolism to steroid hormones, hCG plus AG, or 8-bromo-cAMP plus AG. After 6-48 h of exposure to tropic agents, specific metabolism of [125I]LDL was determined. hCG and 8-bromo-cAMP significantly increased (P less than 0.05) the amount of [125I]LDL bound (2.2-fold), internalized (2.3-fold), and degraded (2.9-fold) by the luteinized granulosa cells. The apparent Km values for LDL degradation in control and hCG-treated cells were similar (2.0 and 2.6 micrograms/ml, respectively). As little as 10 mIU hCG/ml stimulated LDL metabolism in a time-dependent fashion: a stimulatory effect was detected within 6 h of exposure to hCG and was greater after 24 h. AG attenuated but did not prevent the hCG- or 8-bromo-cAMP-stimulated increase in both LDL uptake and metabolism, although it completely inhibited the steroidogenic response. AG alone had no significant effect on [125I] LDL metabolism. We conclude that hCG and cAMP increase LDL metabolism by luteinized human granulosa cells. These effects are apparently not simply a consequence of enhanced cellular cholesterol metabolism to steroids.

Perioperative complications arising after transvaginal oocyte retrieval.

OBJECTIVE: To determine the incidence of perioperative morbidity in patients undergoing transvaginal oocyte retrieval and to identify those patients at greatest risk for complications. METHODS: A retrospective analysis was performed on 674 patients of reproductive age who underwent transvaginal retrieval of oocytes for assisted reproduction technologies during a 3-year period. All procedures were performed by surgeons with extensive experience with transvaginal retrieval. Patients were entered into the program with a primary diagnosis of unexplained infertility (16%), endometriosis (32%), pelvic adhesions and/or tubal occlusion (38%), or infertility from male or immunologic factors (14%). RESULTS: Of the 674 patients studied, ten (1.5%) required hospital admission because of perioperative complications. Nine of these patients needed intravenous antibiotics and one required admission and observation for an expanding broad-ligament hematoma. Six of nine women admitted for antibiotic therapy had a history of extensive pelvic adhesions with or without a history of salpingitis. Five of nine patients had a history of salpingitis. In addition, two patients experienced impressive vaginal arterial bleeding during the procedure. CONCLUSION: This study suggests that transvaginal retrieval may not be as innocuous as is often expressed and that the primary factor predisposing to perioperative morbidity is a history of previous pelvic inflammatory disease and/or adnexal adhesions.

Submucosal leiomyomas and infertility.

Seventeen patients with primary (59%) or secondary (41%) infertility and evidence of at least 1 large (greater than or equal to 5 cm) submucosal leiomyoma underwent abdominal myomectomy . In all patients, no other cause of infertility could be found. Following surgery, 2 of the 17 patients elected to use contraception. Thirteen of the 15 patients wishing to establish a pregnancy had 10 or more months of sexual exposure. The overall pregnancy rate was 47%, and 53% of those patients attempting to establish a pregnancy conceived. The corrected pregnancy rate for those with adequate sexual exposure for at least 10 months was 61.5%. Of those women who attained a pregnancy, 63% did so within 11 months after surgery. Only one patient conceived after more than 3 years (38 months). These results suggest that submucosal leiomyomas are a possible cause of infertility and are highly amenable to surgical treatment and subsequent restoration of fertility.

Follicular aspiration and in vitro fertilization associated with pelvic reconstructive surgery.

Between August 1982 and May 1987, 103 patients underwent in vitro fertilization-embryo transfer (IVF-ET) in association with pelvic reconstructive surgery for infertility. Follicular stimulation was induced with clomiphene citrate and laparotomy scheduled day 12 to 15 of the menstrual cycle. Ultrasound measurements of follicular diameter and number of follicles were obtained on the day of human chorionic gonadotropin (hCG) administration, and laparotomy and ovum retrieval performed 36 hours later. Embryo transfer was performed 48 to 72 hours after insemination. Patients were treated postoperatively with intramuscular progesterone. In addition to evaluating the overall pregnancy rate, the outcome of patients having one or more follicles greater than or equal to 1.4 cm in mean diameter (group A) were compared to those in group B (no follicles greater than or equal to 1.4 cm in diameter). The number of oocytes obtained and the fertilization rate and polyspermic fertilization rate were not significantly different between groups; 10.1% of patients in group A conceived but no patient conceived in group B, yielding an overall pregnancy rate of 8.7%. These data suggest that physicians having IVF-ET at their disposal offer patients IVF during pelvic reconstructive surgery.

Simultaneous ectopic pregnancy with intrauterine twin gestations after in vitro fertilization and embryo transfer.

A simultaneous ectopic tubal pregnancy with viable intrauterine twin gestations after IVF-ET of five fertilized eggs is presented. Pelvic ultrasound and serial quantitative hCG levels were not helpful in the diagnosis of the tubal pregnancy. The risk of multiple pregnancies and of concomitant intrauterine and extrauterine gestations increases with transfer of a greater number of embryos. Karyotype of the tubal pregnancy was normal (46,XX).

Fertilization and cleavage after reinsemination of human oocytes in vitro.

The results of reinsemination of 150 morphologically mature oocytes that failed to fertilize within 15 to 20 hours after initial insemination were evaluated. Only 41 oocytes (27.3%) were fertilized after reinsemination, which is significantly lower than the percentage obtained after initial insemination (74%). However, the cleavage rate was not impaired significantly. Polyspermic fertilization was common (17 oocytes, 29.3%). A total of 64 oocytes were transferred. In 18 patients, all of the oocytes that were transferred had been reinseminated; none of these patients conceived. However, of 28 patients who received embryos arising from both initial insemination and reinsemination protocols, 7 conceived (25%). We suggest that the percentage of fertilization is impaired after 22 hours of incubation in vitro and that those oocytes that have fertilized after reinsemination might partly reflect a misjudgment of initial oocyte maturity. Nevertheless, reinsemination is a relatively simple procedure, with no known attributed risk. Therefore, we recommend reinsemination to increase the overall fertilization percentage. Additional studies are needed to establish better methods for assessment of oocyte maturity to evaluate properly the benefit of such reinsemination procedures.

Comparison of ultrasonographic transurethral and laparoscopic guided oocytes retrieval.

Fifty-eight patients who underwent in vitro fertilization (IVF) treatment were divided into two groups. In 28 patients, ultrasonically guided transurethral follicular aspiration was performed under light general anesthesia. In 30 patients, laparoscopic follicular aspiration was performed under general anesthesia. The mean age for both groups was similar. Follicular recruitment was achieved with human menopausal gonadotropin (hMG) or a combination of clomiphene citrate and hMG. In the ultrasonically guided aspiration group, 128 follicles were aspirated, with a 64% recovery rate (83 oocytes) and an 88.1% embryo transfer (ET) rate (67 embryos). Three (10.7%) intrauterine pregnancies were established in this group. In the laparoscopic group, 153 follicles were aspirated, with a 68.6% recovery rate (105 oocytes) and an 86.3% ET rate (82 embryos), with 4 (13.3%) intrauterine pregnancies established. Three patients had both procedures done; however, none conceived. There were no statistically significant differences between the two groups. Ultrasonically guided transurethral follicular aspiration should be considered as an alternative route for oocyte retrieval, especially when laparoscopy is contraindicated or when the ovaries are not accessible.

The zona-free hamster egg penetration assay as a prognostic indicator in a human in vitro fertilization program.

The present study was designed to test the validity of the hamster egg penetration assay as a prognostic indicator of male fertility in 54 patients undergoing in vitro fertilization. Human oocyte fertilization, cleavage, and pregnancy were compared with the results of this bioassay. Good correlation was found between hamster egg penetration and oocyte fertilization. Conversely, a definite lower limit of hamster egg penetration to define absolute male infertility could not be established because human oocyte fertilization, cleavage, and even pregnancy occurred in spite of low hamster egg penetration.

Effect of follicular size on oocyte retrieval, fertilization, cleavage, and embryo quality in in vitro fertilization cycles: a 6-year data collection.

OBJECTIVE: To determine the effect of follicular size, including the size of the leading follicle, on oocyte retrieval, fertilization, cleavage, and embryo quality in IVF cycles based on a large data collection. DESIGN: Retrospective analysis of 1,109 IVF cycles between 1987 and 1993 at the Hospital of the University of Pennsylvania including 606 patients ranging in age from 23 to 49 years. RESULTS: Follicles with a volume < or = 1 mL show a significantly lower oocyte recovery rate than follicles with a volume of > 1 mL. The highest recovery rate (83.5%) was found in follicles with a volume of 3 to 4 mL. Above a follicular volume of 7 mL, the oocyte recovery drops below that observed for follicles between 1 and 7 mL. Fertilization and cleavage rates were also higher in oocytes obtained from follicles > 1 mL compared with follicles < or = 1 mL. Although fertilization rates were fairly stable above volumes of 1 mL, cleavage rates continued to rise to a peak percentage of 92% with volumes between 6 and 7 mL. Leading follicle size did not have an effect on fertilization and cleavage rates of cohort oocytes. Embryo quality was not influenced significantly by follicular volume. CONCLUSION: Based on this evaluation of a large number of follicles, follicular size is a useful indicator of oocyte recovery, fertilization, and cleavage in IVF cycles. For optimal results, the follicular fluid volume in gonadotropin- and hCG-stimulated cycles should be > 1 mL, which corresponds to a follicle diameter of > 12 mm, and not larger than 7 mL (24 mm). For timing of hCG administration, the number of adequate size follicles appears to be more important than the size of the leading follicle(s).

Follicular maturation parameters associated with the failure of oocyte retrieval, fertilization, and cleavage in vitro.

Follicular maturation parameters predictive of decreased success in human in vitro fertilization were evaluated in 104 gonadotropin-stimulated cycles. Falling estradiol (E2) levels correlated with decreased fertilization (1.9 oocyte/cycle) and cleavage (1.4 oocyte/cycle), when compared with cycles with increasing E2 (2.4 and 2.1 oocyte/cycle, respectively). Likewise, polyspermic fertilization was higher in the former group. Falling E2 levels after human chorionic gonadotropin (hCG) administration correlated with similar trends. Delaying hCG (24 to 96 hours) relative to the last dose of gonadotropin did not affect the total number of oocytes fertilized per cycle. However, polyspermic fertilization and cancellation rates were higher. Leading follicles growing beyond 2.3 cm (determined by ultrasound examination) were associated with decreased recovery, fertilization, and cleavage, and increased polyspermic fertilization. We conclude that ultrasonography and E2 monitoring can predict decreased fertilization and cleavage and should also be monitored after hCG administration.

The effect of immunoglobulin occurring on human sperm in vivo on the human sperm/hamster ova penetration assay.

We selected 91 infertile men who were tested for increased sperm-associated immunoglobulin and also tested in the human sperm/hamster ova penetration assay. There was a statistically significant association between the presence of increased sperm-associated IgG alone (p = 0.0218) and both sperm-associated IgG and A (p = 0.0187) when correlated with the failure to penetrate any hamster ova. There was a trend but no statistical significance when sperm-associated immunoglobulin A alone was present. There was a trend but no statistical relationship between the presence of sperm-associated immunoglobulin and the sperm penetration assay when the criteria for normality of the sperm penetration assay was a 15% or greater ovum penetration rate.

Perinatal outcome of singleton pregnancies conceived by in vitro fertilization: a controlled study.

To determine whether conception by in vitro fertilization and embryo transfer (IVF) predisposes to perinatal complications, the obstetric records of 54 women delivered of singleton pregnancies after conception by IVF were examined. Control women were matched for age, parity, race, year of delivery, diethylstilbestrol exposure and medical problems; another group of women who conceived after infertility treatment was matched in similar fashion. IVF patients showed a longer first stage of labor than previously infertile women, experienced a greater intrapartum blood loss than control or previously infertile women, and showed a trend toward a higher cesarean delivery rate than control women. The differences noted probably do not arise from the physiology of IVF, and although some differences are statistically significant, they are of minimal clinical significance. Singleton pregnancies arising after IVF should not be considered as high risk in the absence of other predisposing factors.

Regulation of cholesterol acquisition and utilization in the corpus luteum.

In this report we have focused on two aspects of control of corpus luteum function; the regulation of lipoprotein uptake and the modulation of cholesterol side chain cleavage through alterations in the phospholipid milieu. Until recently, a role of gonadotropins in the acquisition of lipoprotein-carried cholesterol had not been generally appreciated. However, it is likely that the regulation expression of cell surface lipoprotein receptors will prove to be an important function of tropic hormones, particularly LH, since luteal cells generate progestins at a substantial rate and must, therefore, have an equally substantial supply of cholesterol. Factors other than gonadotropins may have a function in control of lipoprotein receptors and it would not be surprising if luteolytic factors, such as prostaglandin F2 alpha, are found to diminish lipoprotein-uptake as part of their action in reducing steroidogenesis. The control of mitochondrial pregnenolone synthesis by gonadotropin-induced alterations in phospholipid composition of mitochondrial membranes represents a novel concept. Whether certain polar lipids will emerge with the status of intracellular messengers remains to be seen. In any event, our observations clearly demonstrate the possibility for such a mechanism of control.

Uterine arteriovenous malformation in a patient with recurrent pregnancy loss and a bicornuate uterus. A case report.

BACKGROUND: Arteriovenous malformations (AVM) of the uterus are a rare but potential cause of recurrent pregnancy loss. Only four cases of uterine AVM have resulted in a live birth after conservative management. There is no previous report in which a combination of a müllerian anomaly and an AVM existed concomitantly. CASE: A 33-year-old woman with a history of recurrent pregnancy loss was found to have a coexistent uterine AVM and a bicornuate uterus. The patient underwent arterial embolization and Strassman metroplasty and subsequently had a term pregnancy with a live birth. CONCLUSION: Management of uterine AVM should be individualized, taking into account the patient's desire for future fertility and the stability of her health at presentation.

Effects of prolactin on progestin secretion by human granulosa cells in culture.

Concentrations of human prolactin (hPrl) greater than or equal to 600 ng/ml produced inhibition of progestin production in cultures of granulosa cells pooled from follicles of women stimulated with clomiphene citrate-human chorionic gonadotropin (hCG). However, cells collected from follicles of human menopausal gonadotropin (HMG)-hCG-treated patients did not demonstrate a significant reduction in progestin secretion in response to hPrl. We conclude that high concentrations of hPrl can result in inhibition of steroidogenesis, but the expression of the inhibitory effects of Prl depends upon the hormonal treatments used to stimulate follicular growth.

Emotional and psychosocial factors in follow-up of women after IVF-ET treatment. A pilot investigation.

One hundred and fifty-six women who enrolled for treatment in an in vitro fertilization (IVF-ET) program were interviewed, 15 months (mean interval) after the last program contact. Perceptions of treatment stress, decisions about further treatment and the extent of resolution of the infertility crisis were investigated. Standard self-report instruments were used to assess emotional status, self-esteem and marital adjustment. The results showed that only about half the couples who did not achieve pregnancy had decided to terminate treatment. Resolution of infertility was significantly correlated with coping with infertility and with the decision to abandon treatment. Although most patients described treatment as extremely stressful, this did not itself result in emotional distress or dysfunction following treatment, and psychiatric syndromes were infrequent. A deeper understanding of the interactions of these identified factors would help all couples who confront unwanted infertility.