Comparison of different regimens of a gonadotropin-releasing hormone analog during ovarian stimulation for in vitro fertilization.

Three treatment protocols were used in 156 in vitro fertilization cycles. Leuprolide acetate was begun on day 1 of the cycle in one group (n = 20), on day 3 in another (n = 48), and the third control group (n = 88) did not receive the gonadotropin-releasing hormone analog. Human menopausal gonadotropin was initiated on day 3 in all groups. Peak estradiol (E2) levels and the mean numbers of mature oocytes and embryos transferred per cycle were significantly greater in the day 3 group than in either the day 1 or control groups. Patients who received the day 3 protocol had significantly fewer cancelled cycles. A decline in E2 was observed on the third day of analog administration in certain patients, particularly those on the day 1 protocol. Follicle-stimulating hormone and luteinizing hormone (LH) levels increased two- to fivefold 24 hours after initiation of the analog. Thereafter the gonadotropin levels fell, but nevertheless remained above those of controls for most of the cycle. Hence, it appears that enhanced follicular growth attributed to the early transient rises in gonadotropins can be coupled to a suppression of endogenous LH surges in leuprolide-treated women. These beneficial effects seem to be more likely to occur if leuprolide is initiated on cycle day 3 rather than day 1.

Bovine serum albumin (BSA) can replace patient serum as a protein source in an in vitro fertilization (IVF) program.

Alternate protein sources have been suggested to replace the commonly used cord or patient serum for in vitro fertilization (IVF) procedures. During an 11-month period 127 patients treated for in vitro fertilization had either their serum (N = 71) or bovine serum albumin (BSA; N = 56) used as the protein source in the insemination and growth media. Ham's F-10 + 0.5% BSA was used for sperm swim-up and insemination media and 1% BSA was used for the growth media. Patient's serum was added to Ham's F-10 culture media at concentrations of 7.5 and 15% for insemination and growth, respectively. Embryo transfer was performed with Ham's F-10 containing 90% maternal serum in both groups. Fertilization rate of 259 oocytes inseminated in medium containing patient's serum did not differ when compared with 200 oocytes inseminated in medium containing BSA. Likewise, rates of abnormal fertilization, cleavage, and pregnancy were similar in both groups. In a second experiment, 148 normally fertilized oocytes were transferred after 24 hr in culture to growth media containing two different concentrations of BSA (0.5 or 1%). Cleavage rates for the two groups were similar and the percentage of embryos developed to greater than or equal to 4 cells did not differ significantly. We conclude that a single concentration of BSA can safely be used to supplement culture media in human IVF with several practical and economical benefits.

Cortisol levels in human follicular fluid.

This study shows that cortisol levels in follicular fluids in stimulated cycles were correlated with oocyte maturity and in vitro fertilizability. The levels were significantly higher than the concentrations found in spontaneous cycles. Our findings suggest that the presence of cortisol in follicular fluid may play a role in follicular development and oocyte maturation.

Collagen matrix influences the morphologic features and steroid secretion of human granulosa cells.

Steroid secretion and structure of granulosa cells on floating collagen gels were compared with those of cells grown on plastic. Granulosa cells from follicles of gonadotropin-treated women were plated either onto dishes coated with type I collagen or onto plastic dishes. Medium containing serum was removed after 24 hours, defined medium was added, and the gel was floated. Medium was changed daily for 3 days, after which the granulosa cells were prepared for light and electron microscopy. Cells grown on collagen secreted significantly more estradiol and progesterone than those grown on plastic during the 3 days of culture. The round multilayered granulosa cells on collagen had abundant mitochondria and lipid droplets and they formed numerous intercellular junctions. On plastic surfaces, flat granulosa cells grew as a monolayer with few junctions and less abundant mitochondria or lipid droplets. We conclude that growth on floating collagen promotes structural changes of human granulosa cells that enhances cell interaction and secretion of steroid hormones.

An increased initial follicle-stimulating hormone/luteinizing hormone ratio does not affect ovarian responses and the outcomes of in vitro fertilization.

The tenet that a combination of human follicle-stimulating hormone (hFSH)/human menopausal gonadotropin (hMG) improves follicular recruitment was assessed by randomly treating ovulatory women either with hFSH/hMG on days 3 and 4 of the cycle followed by two ampules of hMG daily or with a constant daily dose of 2 ampules of hMG. Estradiol (E2) levels on the day of human chorionic gonadotropin (hCG) and the mean number of mature, immature and atretic oocytes per cycle did not differ between the two groups. Likewise, fertilization, cleavage, and pregnancy rates were similar for the two treatments. When daily hormone levels were compared in 11 patients during two successive treatment cycles with both stimulation protocols, the temporal pattern of FSH accumulation was repeated in both cycles, but FSH levels were significantly higher when patients received hFSH/hMG. Nevertheless, during both cycles, E2 reached similar peak levels and the mean number of follicles per cycle on the day of hCG administration was not different. We conclude that routine use of hFSH/hMG does not improve the success of an in vitro fertilization (IVF) program and that higher FSH levels do not change the individuality of ovarian response in the same woman.

Ovarian response to human menopausal gonadotropin following suppression with oral contraceptives.

The main conclusion of this study is that a profound suppression of the pituitary and ovary can be associated with an inadequate response which may require a longer or different regimen of stimulation to achieve the desired outcome for IVF. We suggest that a pretreatment determination of E2 and gonadotropins can be of value to predict the nature of ovarian response in women with suppressed pituitary-ovarian function.

PIP: A profound suppression of the pituitary and the ovary is connected to an inadequate response to gonadotropin therapy. The therapy, used to produce multiple follicular development for in vitro fertilization (IVF), can cause a variety of responses including: premature luteinization, inadequate amount of preovulatory follicles and an asynchrony of follicular maturation. The aforementioned responses are related to lowered IVF success. IVF patients who received oral contraceptives as part of treatment had undesirable responses to human menopausal gonadotropin when the contraceptives were administered for only a short period. It is suggested that a different regimen be pursued to achieve adequate responses. A pretreatment determination of E and gonadotropins is a viable method of predicting ovarian response among women who have suppressed pituitary-ovarian function. It is further concluded that a favorable response to hMG was not found in IVF patients when treatments of oral contraceptives are administered for a short period.

Flow cytofluorometric analysis of granulosa cell proliferation in rats.

Immature rats were given PMSG followed by an ovulatory dose of hCG. Mature cyclic rats were given an ovulatory dose of hCG on the day of dioestrus, 24 h before the endogenous LH surge was expected to occur. Granulosa cells from individual follicles were subjected to flow cytometry at various times after hormone administration. Administration of PMSG to immature rats caused a marked, transient increase in 'cycling' granulosa cells (cells in S, G2, or M phases of the cell cycle). Administration of hCG to adult cyclic rats caused a slight, but significant, increase in 'cycling' granulosa cells. The results could be taken to indicate that hCG-induced luteinization is not, in fact, accompanied by cessation of cellular proliferation. However, the data lend themselves to alternative explanations. The observed phase shifts may be due to the lengthening of the duration of S and G2 phases, indicating an actual decrease in the rate of cell proliferation. Flow cytometry was found to be a rapid, inexpensive tool for the study of follicular growth in the rat, but, due to variation between follicles and the need for a minimal sample size of 2000 cells, its application is limited to large follicles.

Ovarian response of individuals to different doses of human menopausal gonadotropin.

Hormonal profiles were compared in 14 ovulatory women who were treated with two different doses of gonadotropins in successive in vitro fertilization cycles. All patients suffered from mechanical causes of infertility. Serum estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and progesterone (P) were measured daily during the follicular phase. Women were arbitrarily classified as high responders (E2 greater than 1000 pg/ml on the day of human chorionic gonadotropin administration, n = 8) or as low responders (E2 less than 1000 pg/ml, n = 6), according to the peak E2 levels during the cycle when they received 3 ampules of human menopausal gonadotropin (hMG). When patients were treated with 3 ampules of hMG, serum FSH, LH, and P concentrations increased significantly during the follicular phase in high responders but remained unchanged in low responders. When these patients were treated with 2 ampules of hMG, the temporal profiles of the hormones were similar, but the magnitude of increases in serum levels of gonadotropins and sex steroids was significantly reduced in high responders. The authors conclude that temporal individuality of endocrine profiles cannot be altered by varying the dose of gonadotropin. Increases in hormone levels accompanying a high response to hMG can, however, be dampened by lowering the dose. In contrast, hormone concentrations are not influenced by changing the dose of hMG in low responders.

Cortisol stimulation of estradiol and progesterone secretion by human granulosa cells is independent of follicle-stimulating hormone effects.

The effects of cortisol on steroid secretion by human luteinized granulosa cells was investigated by culturing cells from mature follicles of gonadotropin-treated women. After culturing for 24 hours in medium containing 10% fetal bovine serum, the cells were washed and incubated in a chemically defined medium. Cortisol (10(-5), 10(-6), and 10(-7) M), follicle-stimulating hormone (FSH; 100 ng/ml), FSH plus cortisol (10(-6], or vehicle were added to replicate cultures. After incubating for 6 hours, medium was harvested and assayed for estradiol (E2) and progesterone (P). Cortisol (10(-5), 10(-6) but not 10(-7) M) significantly (P less than 0.05) stimulated E2 secretion when compared to controls. FSH alone did not alter E2 secretion while FSH plus cortisol had a similar effect to cortisol alone. Likewise, P secretion was significantly (P less than 0.05) increased by cortisol when compared with controls and the stimulation was dose dependent. FSH also stimulated P secretion, but the combination of FSH and cortisol did not give an additive or synergistic effect. It appears that cortisol can stimulate the secretion of E2 and P by human granulosa cells without involving FSH receptor interaction. The findings suggest that, in addition to pituitary and ovarian hormones, cortisol may also be directly involved in granulosa cell function.

Incidence of abortion in pregnancies after in vitro fertilization and embryo transfer.

Our purpose was to assess factors that are associated with an increased rate of spontaneous abortion in pregnancies initiated by in vitro fertilization. Pregnancies were diagnosed by measurement of serum human chorionic gonadotropin (hCG) 15 days after embryo transfer. Of the 64 women who conceived, 47 delivered term infants, one patient delivered a stillborn at 22 weeks, 14 aborted in the first trimester, and two had pregnancies that implanted in the tube. Abortion rates were similar for women treated with human menopausal gonadotropin (24%; 12 of 54) and those who received clomiphene citrate (12.5%; one of eight). Two patients conceived after treatment with a combination of clomiphene citrate and human menopausal gonadotropin, neither of whom aborted. In 54 patients treated with human menopausal gonadotropin, there were no significant differences in mean maternal age, number of years of infertility before the pregnancy, history of previous pregnancies, amount of human menopausal gonadotropin used to induce ovulation, serum estradiol levels on the day of hCG administration, mean number of follicles, and the mean number of transferred embryos between the group who delivered and the group who aborted. We conclude that none of these factors are associated with increased tendency for fetal loss in our in vitro fertilization program. Beta-hCG levels on day 15 after embryo transfer were significantly lower in the group who aborted than in the group who delivered, and may be predictive of implantation failure.

Secretory dynamics of bioactive and immunoreactive prolactin in polycystic ovary syndrome.

To further investigate prolactin (PRL) secretion in polycystic ovary syndrome (PCO), the authors evaluated immunoreactive (immuno) and bioactive (bio) PRL levels in the basal state and in response to provocative testing with intravenous dopamine (DA), metoclopramide (MCP), and gonadotropin-releasing hormone (GnRH), before and after disulfiram. Basal measurements of immuno-PRL, bio-PRL, and the ratio of bio/immuno-PRL were similar in PCO and controls. The immuno-PRL decrement after DA was greater than that of bio-PRL in both groups (P less than 0.05). After MCP, immuno-PRL increased more than bio-PRL in PCO (P less than 0.01), and this immuno-PRL increment was greater than that of controls (P less than 0.05). Bio-PRL and immuno-PRL increased after GnRH in PCO, but not controls, and these responses were inhibited by disulfiram. These data confirm PRL hypersecretion in some women with PCO, which is better expressed by immunoreactivity than bioactivity. Given the assay systems and patients studied, bioactivity

Dose of human menopausal gonadotropin influences the outcome of an in vitro fertilization program.

This study compares outcomes of in vitro fertilization (IVF) in two groups of 57 patients when either 2 (group 1) or 3 (group 2) ampules of human menopausal gonadotropin (hMG) were administered daily. Treatment began on day 3 of the cycle and was discontinued when at least 2 follicles attained diameters greater than or equal to 1.5 cm. Human chorionic gonadotropin (hCG) was given either 24 or 48 hours after the last dose of hMG. Although serum estradiol levels were lower in group 1, the average number of oocytes retrieved (3.2 versus 2.9), fertilized (1.9 versus 2.0), and cleaved (1.7 versus 1.8) per completed cycle did not differ between groups 1 and 2. Likewise, the number of oocytes that fertilized abnormally was similar in both groups (0.5 versus 0.3/cycle). However, the number of atretic oocytes (0.03 versus 0.5/cycle) and the percent of oocytes recovered from the cul-de-sac (0 versus 7.2%) were significantly (P less than 0.05) lower in group 1. In group 1, administration of hCG 48 hours after the last dose of hMG was associated with a higher number of cleaving embryos (2.1 versus 1.5/cycle) and a higher pregnancy rate (34.8 versus 14.7%; P less than 0.05) when compared with injection at 24 hours. In group 2, the interval between hMG and hCG did not influence these results. Together, the associations between fewer oocytes that were atretic or recovered from the cul-de-sac, and a trend toward a higher pregnancy rate, suggest that follicular recruitment with 2 ampules of hMG is more appropriate than 3 ampules in an IVF program.

Flow cytometric analysis of deoxyribonucleic acid in human granulosa cells from in vitro fertilization cycles: relationships to oocyte maturity and fertilizability and to follicular fluid steroids.

We measured the mitotic activity of granulosa cells, sex steroid concentrations in follicular fluids, and the maturity and fertilizability of oocytes from 49 follicles. Flow cytometric measurements of DNA were used to determine the percentage of cells in G0/G1, S, and G2/M phases of the cell cycle. Mitotic index was designated as the percentage of granulosa cells in S + G2/M. The progesterone concentration and the progesterone to estradiol ratio in follicular fluids were inversely correlated to mitotic index (r = -0.506; P less than 0.001, and r = -0.320; P less than 0.02, respectively). Estradiol and androstenedione levels did not correlate with the mitotic index. The mitotic index was higher in follicles with immature oocytes [25.6 +/- 2.0% (+/- SE); n = 7] than in follicles with mature oocytes (15.6 +/- 1.2%; n = 41; P less than 0.001). The mitotic index of granulosa cells was lowest in follicles with oocytes that fertilized (15.5 +/- 1.8%), higher in follicles with oocytes that remained unfertilized (18.5 +/- 1.3%; P less than 0.03), and highest in follicles with oocytes that fertilized abnormally (24.0 +/- 2.1%; P less than 0.02). Differences in maturity or fertilizability of oocytes were not associated with variations in follicular fluid progesterone concentrations. The study supports the concept that mitotic activity is decreased when granulosa cells become luteinized. During early follicular growth it is assumed that estradiol and perhaps androstenedione may be important regulators of cell division. Our findings suggest that progesterone, perhaps acting as an antiestradiol, is more important in controlling granulosa cell division of preovulatory follicles during the late follicular phase.

Changes in thyroid function tests and sex hormone binding globulin associated with treatment by gonadotropin.

Increasing levels of E2 in gonadotropin-treated women stimulated hepatic synthesis of SHBG and TBG, and consequently increases in T4 concentration. Nevertheless, unchanged FTI and TSH suggested that a euthyroid state was maintained. The temporal patterns for the rise in serum concentrations of TBG and SHBG during gonadotropin therapy suggest that the synthesis of these proteins by the liver has different sensitivities to E2.

Relationship of human oocyte maturity, fertilization, and cleavage to follicular fluid prolactin and steroids.

Follicular fluid (FF) levels of prolactin (PRL), estradiol (E2), progesterone (P), and androstenedione (delta 4A) were related to diversities in oocyte maturation, fertilization, and cleavage among oocytes obtained for in vitro fertilization. Follicles with mature stage I oocytes contained similar concentrations of PRL and E2, more P, and less delta 4A compared to follicles containing immature oocytes. Follicles with mature stage II oocytes had higher amounts of P and E2 but similar levels of PRL and delta 4A compared to follicles with mature stage I oocytes. Prolactin was lower and E2 higher in FF associated with fertilizable oocytes than in those with unfertilized gametes. Follicles which yielded gametes that ultimately divided to more than four cells had the highest E2 levels, while the lowest E2 concentrations were associated with fertilized, uncleaved eggs. P, delta 4A, and PRL levels in FF were not related to oocyte cleavage. We conclude that human menopausal gonadotropin (hMG) treatment does not necessarily lead to a synchrony or uniformity of preovulatory follicles, maturation of oocytes is associated with rising levels of progesterone and decreasing levels of delta 4A in FF, an increased cleavage rate in vitro is associated with higher FF levels of E2, and high levels of PRL in FF are associated with unfertilized oocytes. These findings emphasize that differences in the hormonal milieu surrounding oocytes may have profound effects on the success of in vitro fertilization.

Relationships between polypronuclear fertilization and follicular fluid hormones in gonadotropin-treated women.

Similar incidences of congenital abnormalities in in vitro fertilization and embryo transfer (IVF-ET) pregnancies and births in the general population have not supported earlier concerns that IVF may increase chromosomal aberrations. Nevertheless, polypronuclear fertilization is a common, undesirable, and poorly understood outcome of IVF. We evaluated hormone levels in 20 follicular fluids that were associated with mature oocytes that have fertilized abnormally (greater than or equal to pronuclei) and compared them to follicles with mature oocytes that cleaved normally, fertilized but failed to cleave, or did not fertilize. Progesterone (P), androstenedione, estradiol, percent free estradiol, sex hormone-binding globulin, insulin, and prolactin were measured. P levels were significantly higher in follicular fluids associated with oocytes that fertilized abnormally than in the other groups. Levels of the other hormones were similar in all the study groups. We conclude that IVF of oocytes from highly luteinized follicles as judged by P levels may result in polypronuclear fertilization.

Prostaglandin F2 alpha treatment of canine pyometra.

Immediate and long-term outcomes of prostaglandin F2 alpha treatment for canine pyometra were studied in 10 bitches. Examination of pretreatment uterine biopsy specimens, taken for histopathologic diagnosis and classification of disease severity, revealed either type III or IV pyometra. Dinoprost tromethamine (0.25 or 0.5 mg/kg of body weight, SC) was given once daily for 3 days. Bitches were bred at the first posttreatment estrus and monitored for a minimum of one year. When pure cultures of Escherichia coli (n = 3) or Staphylococcus aureus (n = 1) were obtained from the vagina, these bacteria also were found in the uterus. Pretreatment WBC counts often did not reflect the severity of histopathologic findings in the uterus, but posttreatment WBC counts were useful in monitoring response to treatment. Four bitches produced a litter within one year of treatment. Four bitches (40%) had recurrence of pyometra within one year of treatment, and these same bitches had another recurrence after an additional prostaglandin treatment. Three additional bitches had a recurrence by 27 months after therapy, establishing a total recurrence rate of 77% (7/9). Results suggested that subclinical disease may persist after treatment, with clinical recurrence during diestrus. Despite the high recurrence rate, it was concluded that this treatment is a practical treatment for canine pyometra when reproduction is desired.

Total estradiol, free estradiol, sex hormone-binding globulin, and the fraction of estradiol bound to sex hormone-binding globulin in human follicular fluid.

Sex hormone-binding globulin (SHBG), percent free estradiol (E2), the fraction of E2 bound to SHBG, and total E2 were measured in the serum and follicular fluid of 12 women (25 follicles) who had received gonadotropin stimulation in an in vitro fertilization program. The women were classified as high or low responders based on peak serum E2 levels (high responders: peak E2, greater than 1500 pg/ml; low responders: peak E2, less than 1000 pg/ml). During treatment, serum levels of SHBG increased in high responders from 55 +/- 8.8 (+/- SEM) to 96 +/- 16 nM (P less than 0.01), but did not change in low responders. SHBG was more concentrated in follicular fluids from high responders (142 +/- 12.5 nM) than in those from low responders (44.4 +/- 5.8 nM). A positive correlation was found between serum and follicular fluid levels of SHBG (r = 0.873; P less than 0.01). In follicular fluid, total E2 levels, which varied from 100-2650 ng/ml, correlated (r = 0.790; P less than 0.01) closely with SHBG levels. The percent free E2 averaged 5.9% (range, 4-10.6%) in follicular fluid compared to 1.8% (range, 1.5-2.1%) in serum. An inverse correlation (r = -0.661; P less than 0.01) was found between total E2 concentrations and percent free E2 in follicular fluid. The relationship between serum and follicular fluid levels of SHBG suggests that SHBG in follicles arises from the circulation. Although SHBG is present in follicular fluid in amounts similar to those in serum, the large quantities of E2 in preovulatory follicules exceed the binding capacity for SHBG, and the majority of E2 appears to be bound to albumin. Hence, it seems unlikely that SHBG in follicular fluid regulates estrogen action in ovarian target cells.

Changes in serum sex hormone-binding globulin, free estradiol, and testosterone during gonadotropin treatment.

Sex hormone-binding globulin (SHBG), estradiol (E2), percent free E2, percent of E2 bound to SHBG, and testosterone (T) were evaluated in 28 ovulatory women during human menopausal gonadotropin-stimulated cycles for in vitro fertilization. Patients were divided into two categories: low responders, in whom serum E2 concentration reached levels less than 1000 pg/ml (mean, 638 +/- 93), and high responders, with serum E2 levels greater than 1000 pg/ml (mean, 2219 +/- 330). A significant increase in SHBG can occur within a short time in high responders (from 62.8 to 103.9 nmol/l) but not in low responders. This increase is accompanied by a significant decrease in the percent free (bioavailable) E2, but the distribution of E2 between the fraction bound to SHBG or albumin did not vary. Despite the increase in the levels of SHBG, the concentration of bioavailable (free) E2 in hyperstimulated women is higher than in normal cycles. The significant increase in T in high responders, by virtue of its higher affinity for SHBG, probably contributes to the increased levels of bioavailable E2.