Initiation of angiogenesis by human follicular fluid.

Angiogenesis was observed and measured after injection of human follicular fluid into rabbit corneas. Undiluted human follicular fluid stimulated angiogenesis in every case, with new blood vessels visible 3 days after injection and extending 2.0 millimeters from the corneal scleral limbus into the injection site by day 15. Stimulation of angiogenesis was lost by heating or diluting the follicular fluid but was retained after charcoal stripping or dialysis. Human follicular fluid contains an angiogenic factor that may be associated with perifollicular neovascularization during folliculogenesis.

Intragonadal regulation of follicular maturation.

Although there are interspecies of variations in the process of follicular development, a generalized summary is presented that encompasses theories of follicular maturation from laboratory and domestic animals, nonhuman primates, and women. As there are many new substances whose actions within the follicle are unknown, it is difficult to ascribe definitive roles to these proteins in follicular development and ovulation. However, where possible, these substances are included in the summary. During early follicular development, FSH binds to granulosa cells of primary follicles to stimulate production of estradiol by the induction or enhancement of aromatase synthetase (37, 336, 337). Estradiol, in turn, induces proliferation of granulosa cells (338-344) and increases the sensitivity of the follicle to further gonadotropin stimulation (12, 339, 345-349). Estradiol can synergize with gonadotropins to increase ovarian weight, enhance proliferation of granulosa cells, and promote growth of preantral follicles and antrum formation (345, 347, 350-352). In addition, estradiol enhanced the responsiveness of granulosa cells to FSH and LH by increasing synthesis of progesterone (353, 354). The generalized enhancement of gonadotropin action by estradiol is partially mediated by FSH-induced accumulation of cAMP. However, as synthesis of estradiol increases, this steroid directly stimulated follicular growth, since estrogens have long been known to stimulate growth of ovarian cells and exert a direct antiatretic effect (355, 356). However, the exact mechanism involved in follicular growth achieving preovulatory status rather than undergoing atresia remains uncertain. Estradiol not only enhances gonadotropin stimulation of LH and FSH receptors in granulosa cells (357, 348) but is required for FSH induction of FSH receptors (359, 360). Estradiol alone can increase numbers of its own receptor in granulosa cells (350) as well as increase its own production by stimulating aromatase activity (361). Estradiol secreted by the dominant follicle has a positive feedback effect on the hypothalamus and pituitary, enhancing gonadotropin secretion and ensuring the preovulatory gonadotropin surges (362). The increased gonadotropins can further increase the production of estradiol which, in turn, enhances its own production. Therefore, estradiol is included in two positive feedback loops (one at the pituitary and one at the ovary) to maintain the dominant follicle and ensure ovulation. Progesterone and androgens also have intrafollicular effects on follicular growth and steroidogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Steroidogenesis of porcine granulosa cells from small and medium-sized follicles: effects of follicle-stimulating hormone, forskolin, and adenosine 3,'5'-cyclic monophosphate versus phorbol ester.

We studied the effects of porcine FSH, forskolin, and (Bu)2cAMP [agents that stimulate steroidogenesis via the adenylate cyclase-cAMP pathway (cAMP system)] either alone or with concomitant addition of phorbol 12-myristate 13-acetate (TPA; a phorbol ester that activates protein kinase-C) on steroidogenesis in porcine granulosa cells cultured from small (less than 3 mm) and medium-sized (3-6 mm) ovarian follicles. We attempted to determine if granulosa cells from different maturational states had different responses to these agonists and antagonists. Cells were cultured in serum-free medium 199 supplemented with insulin (10 micrograms/ml), transferrin ( 5 micrograms/ml), and androstenedione (2.5 X 10(-7) M) for 48 h. Levels of progesterone (P) and estradiol (E2) were determined in spent medium by RIA. We found that FSH, forskolin, and cAMP all stimulated secretion of E2 and P in a dose-dependent manner in both developmental groups. When TPA was added alone to cultures, P levels were stimulated at low doses of TPA but inhibited at higher doses in granulosa from both sized follicles, whereas cells from both small- and medium-sized follicles demonstrated reductions in E2. TPA was also found to inhibit FSH-, forskolin-, and cAMP-induced steroidogenesis in a dose-dependent manner in cells from the two groups of follicles. The stimulatory effects of any of the secretagogues on E2 secretion were inhibited by TPA to a significantly greater extent in granulosa cells from small follicles. Although inhibition of FSH- and forskolin-induced P secretion by TPA was also greater in granulosa cells from small follicles, cAMP-treated cells did not show this differential inhibition. Thus, it appears that modulators of the protein kinase-C system regulate steroidogenesis differently in granulosa cells from small and medium follicles. These differences may involve alterations in the interplay between the protein kinase-C and cAMP pathways.

Stage dependent expression of inhibin alpha and beta-B subunits during the cycle of the rat seminiferous epithelium.

In order to elucidate the putative role of inhibin in regulation of spermatogenesis, expression of inhibin subunits was examined at defined stages of the cycle of the rat seminiferous epithelium. Twenty 2-mm segments of seminiferous tubules at stages XIII-I, II-VI, VII-VIII, and IX-XII were dissected using the transillumination technique and subunit specific messenger RNAs (mRNAs) were quantitated by filter hybridization. The alpha and beta-B subunit mRNAs varied significantly in different stages, the highest levels of both alpha and beta-B subunit expression were seen in stages XIII-I and the lowest in stages VII-VIII. The hybridization signals obtained with beta-actin probe were not significantly different between different stages indicating that the differences in the quantities of subunit mRNAs in different stages were not due to different amounts of RNA blotted. beta-A subunit mRNA levels were below the detection limit of the filter hybridization method. These data demonstrate that expression of inhibin alpha and beta-B subunits in the rat testis is stage dependent and suggest a paracrine role for inhibin-related peptides in regulation of spermatogenesis.

Effect of human menopausal gonadotropin and follicle regulatory protein(s) on 3 beta-hydroxysteroid dehydrogenase in human granulosa cells.

Recently, a protein fraction [follicle regulatory protein (FRP)] which inhibits FSH-induced granulosa cell aromatase activity was isolated from both human and porcine follicular fluid. In this study, the actions of FRP on 3 beta-hydroxysteroid dehydrogenase (3 beta-HSDH) activity were examined using granulosa cells obtained from hyperstimulated patients undergoing oocyte aspiration for in vitro fertilization. Granulosa cells were cultured with 0, 167, or 500 micrograms/ml FRP with or without human menopausal gonadotropin (hMG; 10 mIU/ml). After 48 h, the medium (S) was removed and stored. Cells then were mechanically lysed and centrifuged at 10,000 X g. The supernatant was further centrifuged (100,000 X g) to obtain a microsomal fraction (M) and cytosol (C). The M fraction was resuspended in medium 199 with 10(-6) M pregnenolone plus 5 microM NAD+ and incubated for 2 h to determine 3 beta-o1 dehydrogenase activity. The S, C, and M fractions were all assayed for progesterone (P) by RIA. hMG markedly increased P concentrations in the S and C fractions. The M fraction demonstrated a hMG-dependent enhancement in 3 beta-HSDH activity. However, the hMG-associated S, C, and M P levels were decreased in granulosa cells coincubated with FRP. In conclusion, ovarian steroidogenesis may be dependent on the integrated interactions of both gonadotropins and local nonsteroidal paracrine/autocrine modulators of granulosa cell function.

Fluorescence localization of luteinizing hormone/human chorionic gonadotropin uptake in the primate ovary. II. Changing distribution during selection of the dominant follicle.

Gonadotropin receptors have been localized in ovarian compartments primarily by incorporation of radiolabeled hCG. The recent development of a fluorescein-hCG conjugate retaining biological and immunological properties similar to native hCG has facilitated localization of ovarian LH/hCG binding in vivo. During menses in the primate ovarian cycle, no follicle in either ovary demonstrated a strong affinity for the hormonal conjugate. However, areas of fluorescence was associated with the interstitial cell glands of both ovaries from each monkey throughout the ovarian cycle. By the seventh day of the menstrual cycle, a unique pattern of thecal fluorescence circumscribed a single follicle in one ovary from each monkey. By days 9 and 11, the follicle surrounded by the unique thecal fluorescence was clearly the largest follicle in either ovary, such that it was judged to be the dominant follicle. On the basis of unique binding characteristics of the fluorescein-hCG conjugate by the surrounding thecal tissue, the putative dominant follicle was identifiable by day 7 of the menstrual cycle, even though its size was not yet a distinguishing factor. These findings further suggest that thecal tissue associated with the dominant follicle, and perhaps the interstitial component, influences the evolution of the dominant follicle through enhanced LH uptake during the midfollicular phase of the primate ovarian cycle.

Follicular fluid steroid levels in dysmature and mature follicles from spontaneous and hyperstimulated cycles in normal and anovulatory women.

Follicular maturity and atresia have been defined previously, both hormonally and microscopically, in normal ovulatory women. Ovarian hyperstimulation with clomiphene citrate and human menopausal gonadotropins in normal women is carried out for the purpose of aspirating oocytes from several large follicles for in vitro fertilization. Alterations in follicular fluid (FF) hormone levels occur with hyperstimulation regimens, and some of these large follicles (greater than 18 mm) appear morphologically atretic. We have used the term dysmature to describe those large follicles that have an abnormal oocyte morphological appearance and cannot be fertilized in vitro. Mature follicles have been defined by their size, their oocyte morphological appearance, and their ability to be fertilized in vitro. FF from small (2-3 mm) and large (greater than 18 mm) mature and dysmature follicles were obtained from 10 untreated ovulatory women. Mature and dysmature follicles also were obtained from clomiphene and human menopausal gonadotropin-treated normal (n = 11) and anovulatory (n = 5) women. In untreated cycles, the FF steroid content of the small follicles characterized these follicles to be atretic. FF from dysmature follicles from spontaneous untreated cycles had higher concentrations of dihydrotestosterone, 5 alpha-androstane-3 alpha,17 beta-diol, and 17 beta-estradiol (E2) and lower progesterone (Prog) and Prog to E2 ratios (P less than 0.05). Compared to mature follicles from untreated patients, hyperstimulated mature follicles from ovulatory women had higher FF E2 concentrations and lower Prog to E2 ratios (P less than 0.05). In ovulatory patients, the FF concentrations of testosterone were higher and FF Prog and Prog to E2 ratios were lower (P less than 0.05) in the dysmature than in the mature follicles. Mature follicles from hyperstimulated ovulatory patients and those from hyperstimulated anovulatory patients were similar except for lower FF Prog, higher FF E2, and lower Prog to E2 ratios in the anovulatory group. Dysmature follicles from hyperstimulated anovulatory patients had lower FF androstenedione and dihydrotestosterone, but were generally similar to mature follicles. The percentages of dysmature follicles occurring among all large (greater than 18 mm) follicles that were aspirated were similar in ovulatory (34%) and anovulatory (45%) patients. FF steroid concentrations did not correlate with serum levels of testosterone and E2 at the time of follicle aspiration in any patient group. In conclusion, FF androgen concentrations in hyperstimulated follicles were unrelated to morphological maturity.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of proteins in pooled human follicular fluid which suppress follicular response to gonadotropins.

To evaluate the role of nonsteroidal, follicular fluid proteins in folliculogenesis, the 10-55% saturated ammonium sulfate fraction of pooled human follicular fluid was dialyzed against 0.025 M Tris/HCl (pH 7.5) using 10,000 molecular weight exclusion membranes, then passed through agarose immobilized textile dye. Activity was determined by test fraction inhibition of human menopausal gonadotropin (2 U human LH/FSH . day), induced ovarian weight, and serum estradiol increase in hypophysectomized, diethylstilbesterol-treated, 25-day-old female rats. Specific inhibition (89 +/- 6.8% SEM) of ovarian weight increase was found in the material (2 ml) eluted from an Orange A column with KCl (1.5 M, pH 6.8). Inhibitory activity of the Orange A-bound material, which eluted through a standardized Sephadex G-50 column, corresponded to a molecular weight of 13,000-25,000. Isoelectric focusing on a Sephadex G-15 support bed or ampholyte displacement chromatography of Orange A bound material demonstrated inhibitory activity at pH 3.5-4.5 and 6.5-7.0. No demonstrable activity was found in similar fractions eluted through a Concanavalin A-Sepharose 4B column before or after addition of alpha-methyl-D-mannoside (2 M, pH 7). When active fractions were heated (56 C, 1 h) or exposed to trypsin (10 mg/100 ml), activity was lost. When aliquots of the saturated ammonium sulfate-extracted, dialyzed, Orange A-bound eluent were separated by high performance liquid chromatography using gel exclusion columns, activity in the bioassay was recovered in the 13,000-35,000 molecular weight range. Although confirmatory data await further studies, it is tempting to speculate that this protein(s) may be an important inter- and/or intraovarian regulator of follicular response to gonadotropins.

Anovulation after pregnancy termination: ovarian versus hypothalamic-pituitary factors.

PIP: 28 female rhesus monkeys were studied after induced abortion or after spontaneous delivery at term to differentiate ovarian from hypothalamic-pituitary factors responsible for suppression of ovulation. Human menopausal gonadotropin (HMG) was administered to stimulate follicular maturation, and estradiol benzoate was injected to test induction of gonadotropin surge. Spontaneous ovulation was delayed until about 39 days after abortion. The presence of multiple preovulatory follicles on both ovaries confirmed that folliculogenesis had been stimulated by HMG, which also caused the maturation and rupture of these follicles. These findings show that pregnancy does not hinder gonadotropin stimulation on the ovaries, but that it suppresses hypothalamic-pituitary responsivity to the positive feedback action of estrogen on FSH and on LH surges. It is possible that tonic gonadotropin secretion is inadequate to initiate follicular maturation until late in the puerperium.^ieng

Asymmetrical ovarian function during recruitment and selection of the dominant follicle in the menstrual cycle of the rhesus monkey.

Despite similar exposure to pituitary gonadotropins by perfusion of both ovaries with the same peripheral blood, only 1 of the 2 ovaries sponsors the single dominant follicle in the typical menstrual cycle. In the present study was examined the initiation of asymmetrical ovarian function during recruitment and selection of the dominant follicle in the primate ovarian cycle by comparison of steroid hormones in the ovarian venous effluent. Thirty-four adult female rhesus monkeys were selected because of high estimated fertility based on their reproductive performance. These monkeys underwent laparotomy for ovarian inspection and collection of ovarian venous blood on 1 of days 1, 3, 5, 7, 9, and 11 after the onset of menses. In addition, femoral blood was collected daily. Repeat laparotomies were performed in the midluteal phase to assess the location of the functional corpus luteum. Concentrations of 17 beta-estradiol, androstenedione, and progesterone were determined in all sera, as well as LH and FSH in peripheral sera, by RIA. In all, 17 of 19 ovulatory monkeys manifested clear asymmetry of 17 beta-estradiol 5 days before the LH/FSH midcycle surges. Often, asymmetry of androstenedione levels was not apparent until 3 days before the midcycle gonadotropin surge. Uniformly, in ovulatory monkeys, the ovary associated with significantly greater concentrations of 17 beta-estradiol and androstenedione in ovarian venous serum ultimately bore the functional corpus luteum observed in the midluteal phase and confirmed by elevated progesterone in peripheral serum. We interpret these findings to indicate that asymmetrical ovarian steroid secretion, especially of 17 beta-estradiol, may be among the earliest indicators that the dominant follicle, or at least the ovary destined to bear it, is already selected by 5 days before the preovulatory FSH/LH surge in the typical menstrual cycle.

Correlation of human follicular fluid inhibin activity with spontaneous and induced follicle maturation.

We sought to correlate the inhibin activity of individual ovarian follicles (greater than 16 mm in diameter) from untreated (7 patients; 7 follicles), clomiphene-stimulated (150 mg/day; menstrual cycle days 5-9; 9 patients, 14 follicles), and human menopausal gonadotropin (hMG)-stimulated (150 IU/day; menstrual cycle days 3-11; 8 patients; 23 follicles) ovarian cycles and to correlate these results with the follicular fluid (FF) steroid concentration. Follicular aspirates were obtained via laparoscopy from 24 regularly menstruating patients when the diameter of the largest follicle reached 20 mm, as determined by serial ultrasonography. FF concentrations of estradiol, progesterone, testosterone, 17-hydroxyprogesterone, and androstenedione were determined by RIA. Inhibin activity was determined using the inhibition of basal 24-h FSH secretion by dispersed rat anterior pituitary cells. Inhibin values were highest among the follicles aspirated from those patients who received hMG [277 +/- 31 (+/- SE) U/ml] compared to untreated subjects (51 +/- 13 U/ml) or those who received clomiphene (96 +/- 14 U/ml). Estradiol was highest in FF from untreated patients (2295 +/- 1155 ng/ml) compared to levels in patients who received hMG (368 +/- 1.76 micrograms/ml) or clomiphene (1049 +/- 174 ng/ml). FF progesterone values were highest in untreated patients (9.4 +/- 2.59 micrograms/ml) compared to those in hMG-treated (5.04 +/- 1.76 micrograms/ml) and clomiphene-treated patients (7.82 +/- 1.24 ng/ml). FF 17-hydroxyprogesterone values (7.82 +/- 1.24 ng/ml). FF 17-hydroxyprogesterone values were similarly higher in the untreated (1.55 +/- 0.21 micrograms/ml) and clomiphene-treated (2.54 +/- 0.27 micrograms/ml) patients than in the hMG-treated group (0.73 +/- 0.09 micrograms/ml). FF androstenedione (untreated, 50.7 +/- 30 ng/ml; clomiphene-treated, 73.4 +/- 23.4 ng/ml; hMG-treated, 60.2 +/- 19.8 ng/ml) and testosterone (6.66 +/- 2.45, 5.98 +/- 1.46, and 6.39 +/- 2.16 ng/ml, respectively) concentrations in all three patient groups were similar. In untreated patients, there was a highly significant positive correlation between intrafollicular inhibin activity and FF estradiol, testosterone, and androstenedione concentrations and a statistically significant negative correlation between intrafollicular inhibin activity and FF progesterone concentrations. Patients receiving clomiphene therapy demonstrated at least two different response patterns, one with a positive and one a negative correlation between intrafollicular inhibin activity and FF steroid concentrations. The patients receiving hMG therapy had no statistically significant correlation between intrafollicular inhibin

Ovulation induction in clomiphene-resistant anovulatory women: differential follicular response to purified urinary follicle-stimulating hormone (FSH) versus purified urinary FSH and luteinizing hormone.

We studied 15 anovulatory women undergoing ovulation induction with purified human urinary FSH or purified human urinary FSH and LH [human menopausal gonadotropins (hMG)]. All patients had either sporadic or no vaginal bleeding after progesterone therapy and failed to ovulate after receiving clomiphene (250 mg for 5 days) plus hCG. Other causes of infertility were ruled out. Sixteen cycles of FSH and 12 cycles of hMG were administered according to a standard protocol. Estradiol, progesterone, androstenedione, testosterone, LH, and FSH concentrations were quantitated by RIA. Follicular diameter was determined using ultrasound. There was no significant difference in the amount of FSH or hMG used per patient, in the duration of therapy before hCG administration, or in the length of the luteal phase in any patient. There was a difference in the number of follicles greater than 1000 mm3 per cycle in those patients receiving FSH compared to the number in those receiving hMG [2.8 +/- 1.3 (+/- SEM) vs. 4.4 +/- 1.5 follicles; P = 0.026). The maximum follicular phase serum estradiol (18.3 vs. 34.8 ng/ml) and maximum luteal phase progesterone concentrations (1289 vs. 2808 pg/ml; P = 0.026) were also different between the FSH and hMG groups. Linear regression analysis revealed a significant correlation between the peripheral serum estradiol levels and the total follicular volume of follicles in the hMG-treated group which was not apparent in the FSH-treated group. These findings suggest that exogenous LH may not be required to induce folliculogenesis in anovulatory patients.

Activity of a human follicular fluid protein(s) in spontaneous and induced ovarian cycles.

Recently, we identified a human follicular fluid protein(s) (FP) which inhibited human menopausal gonadotropin (hMG)-induced rat ovarian weight gain and FSH-induced aromatase. Here, we assessed FP activity from ovulatory patients who were either untreated (n = 7) or received clomiphene (n = 9; 150 mg/day on cycle days 5-9) or hMG (n = 6; 150 IU/day on cycle day 3). Aspirations were performed when one follicular diameter exceeded 20 mm. FP activity was expressed as the percent inhibition of porcine granulosa cell aromatase activity at three concentrations of extracted follicular fluid (range, 1250-10 micrograms; extrapolated to 50 micrograms). Patients receiving hMG or clomiphene had multiple follicles greater than 16 mm in diameter (3.83; 2.66/patient, respectively), while untreated patients had 1 each. FP activity was 14.1 +/- 5.3% (mean +/- SEM) inhibition for untreated, 18.0 +/- 3.4% inhibition for hMG-treated, and 13.7 +/- 5.3% inhibition for clomiphene-treated patients. Follicular fluid estradiol levels from untreated patients (2590 +/- 1221 ng/ml) were greater than estradiol concentrations from hMG-treated (356 +/- 55 ng/ml; P less than 0.01) or clomiphene-treated (1317 +/- 344 ng/ml; P less than 0.05) patients. Progesterone follicular fluid levels were 9.84 +/- 3.3, 5.18 +/- 61, and 11.3 +/- 2.3 micrograms/ml for untreated, hMG-treated, and clomiphene-treated patients, respectively (P less than 0.05). A similar relationship was present with 17-hydroxyprogesterone (untreated, 1.6 +/- 0.2 micrograms/ml; hMG-treated, 0.76 +/- 0.1 micrograms/ml; clomiphene-treated, 2.16 +/- 0.3 micrograms/ml; P less than 0.05). Androstenedione and testosterone follicular fluid levels were similar in all groups (78.9 +/- 23 and 7.09 +/- 2.14 ng/ml, respectively). Untreated patients had a positive correlation between FP and follicular fluid estradiol (r = 0.689; P less than 0.01) and inhibin activity (r = 0.654; P less than 0.05), and a negative correlation between follicular fluid progesterone levels (r = 0.622; P less than 0.05). Patients treated with hMG had a significant negative correlation between FP activity and follicular fluid progesterone levels (r = 0.756; P less than 0.005) and a biphasic correlation with follicular fluid 17-hydroxyprogesterone (r2 = 0.853; P less than 0.0025). Clomiphene-treated patients had biphasic correlations between follicular fluid estradiol and 17-hydroxyprogesterone levels (r2 = 0.853 and P less than 0.0025, and r2 = 0.637 and P less than 0.025, respectively). These findings indicate that the FP activity of the dominant follicle correlates with its state of differentiation, as described by intrafollicular estradiol, progesterone, 17-hydroxyprogesterone levels and inhibin activity. These relationships are in part dependent upon gonadotropin stimulation.

Ibuprofen in the prevention of experimentally induced postoperative adhesions.

Using a five-dose 70 mg/kg regimen, of ibuprofen significantly reduced both overall adhesion formation and the severity of adhesions when given to rabbits in the immediate postoperative period. This reduction was apparently time- and dose-related, as animals given smaller doses of ibuprofen at subsequent intervals had a tendency toward more adhesions and also more severe adhesions. A positive correlation was found between the severity of adhesions and the formation of new glycosaminoglycans and collagens.

Localization of follicle regulatory protein in the porcine ovary.

The granulosa cell secretes a protein (follicle regulatory protein: FRP) that affects the responsiveness of other follicles to gonadotropin stimulation. This protein was purified, partially characterized, and rabbit antisera as well as monoclonal antibodies were prepared against FRP. Fixed sections of porcine ovaries were prepared on slides and then incubated with the monoclonal antibody or polyclonal antisera and then incubated with either biotinylated mouse IgM or rabbit IgG antisera, respectively. These sections were then incubated with avidin conjugated to horseradish peroxidase, followed by substrate. Staining with both the monoclonal antibody and the antisera was present in the cytoplasm of granulosa cells of small- or medium-sized antral follicles. Staining distribution was localized preferentially to cells near the basal lamina; the antral granulosa cells of viable follicles did not stain. Neither primordial follicles nor pre-antral follicles (less than 300 microns in diameter) showed any positive staining. Thecal cells were not stained in follicles less than 5 mm in diameter, whereas some large follicles (greater than 5 mm) contained staining in the theca. In the latter, specific granulosa staining was only weakly positive with the polyclonal antibody and negative with the monoclonal antibody. Atretic follicles contained significant staining of all epithelial cells adjacent to the basal lamina by both the monoclonal and polyclonal antibody preparations. Staining of the luteal ovary by the monoclonal antibody was limited to the large luteal cells. These findings suggest that FRP is produced by the granulosa cells of porcine follicles at the stage of maturation corresponding to 0.5 mm in diameter. As the viable follicle increases in size, production of FRP in the granulosa is reduced below the detectable level when the follicle exceeds 5 mm in diameter. The main source of FRP during the luteal phase is the large cell of the corpus luteum.

Physiologic estradiol replacement following oophorectomy: failure to maintain precastration gonadotropin levels.

To determine whether regularly menstruating women retain an intact pituitary sensitivity to estrogen negative feedback following bilateral salpingo-oophorectomy, 6 parous women, aged 31 to 47, reporting regular menstrual cycles were studied following total abdominal hysterectomy and bilateral salpingo-oophorectomy for non-endocrine-related indications. During closure of the incision, a 12.5-mg continuous-release crystalline 17 beta-estradiol pellet was implanted in the subcutaneous tissue. Antecubital venous blood was sampled preoperatively and at 1, 4, and 8 weeks postoperatively and stored frozen (-15C) until assayed for follicle-stimulating hormone (FSH), luteinizing hormone (LH), and 17 beta-estradiol by radioimmunoassay. Despite maintenance of serum estradiol levels in the mid-follicular phase range (40 to 80 pg/ml), the serum concentration of both FSH and LH progressively increased. By the fourth postoperative week, both FSH and LH concentrations had increased significantly (P less than .05). This trend continued and, by 8 weeks after surgery, both gonadotropin levels were in the postmenopausal range. The authors conclude that even when replacement begins immediately following castration, physiologic estradiol at concentrations characteristic of the normal midfollicular phase is not sufficient to maintain suppression of gonadotropins. Whether these observations reflect a change in pituitary sensitivity to 17 beta-estradiol feedback, the removal of nonsteroidal inhibitory substances of gonadal origin, or the lack of an appropriate composite steroidal milieu remains unanswered.

Inhibition of ovarian cancer cell proliferation in vivo and incorporation of 3H-thymidine in vitro after follicle regulatory protein administration.

Follicle regulatory protein immunoreactivity and biologic activity were measured in ascites from a patient with juvenile granulosa cell tumor. Microscopic examination of immunohistochemical staining of a juvenile granulosa cell tumor with anti-follicle regulatory protein antisera showed homogeneous cytosolic expression of follicle regulatory protein throughout the tumor. Tumor cells were injected subcutaneously into nude mice. Partially purified follicle regulatory protein (50 micrograms/day) was then injected daily for 10 days, or for 25 days once the tumor became palpable. Treatment with follicle regulatory protein significantly slowed the rate of tumor growth with both treatments. To test the tissue specificity of the effect, a metastatic, well-differentiated endometrial adenocarcinoma was also grown in nude mice. Follicle regulatory protein treatment did not alter the rate of tumor growth. An in vitro clonigenic assay confirmed these in vivo results. Partially purified follicle regulatory protein had a biphasic effect on the proliferation of juvenile granulosa tumor cell but did not affect the proliferation of endometrial adenocarcinoma cells. Clonigenic assays were performed on five ovarian adenocarcinomas passaged in vitro, and these tumor cells exhibited a biphasic response to follicle regulatory protein. Immunoneutralization studies showed that this biphasic response was due to impurities in the follicle regulatory protein preparations. The longer the exposure of the tumor cells to follicle regulatory protein, the greater the degree of inhibition of proliferation. In summary, administration of follicle regulatory protein slowed tumor growth through a direct effect on the tumor cell rather than an indirect effect on the hormonal or immune status of the host.

Alteration of follicle regulatory protein levels in human reproductive disorders: anovulation.

Recently, a follicle regulatory protein was identified that suppresses ovarian response to gonadotropins. In this study, the serum levels of follicle regulatory protein were measured in five groups of women: reproductive age undergoing oophorectomy (N = 10), postmenopausal (N = 10), ovulatory (N = 13), anovulatory (N = 16), and anovulatory receiving clomiphene citrate therapy (N = 14). Follicle regulatory protein-related immunoreactivity was measured by a competitive enzyme-linked immunosorbent assay, while peripheral estradiol and progesterone levels were determined by established radioimmunoassay. Concentration of follicle regulatory protein in serum in all ovariectomized patients decreased significantly from preoperative levels. The postmenopausal women had significantly lower follicle regulatory protein levels than did ovulatory and anovulatory women. Patients with low levels of serum estradiol in the early follicular phase exhibited either significantly elevated or suppressed follicle regulatory protein levels compared with patients with normal estradiol concentrations, suggesting two different etiologies for ovarian dysfunction. Eleven to 12 and 22-23 days after the onset of the last menstrual period, patients with elevated follicle regulatory protein levels were found to be anovulatory. These observations suggest that elevated intraovarian levels of follicle regulatory protein may cause a disruption of follicular maturation that leads to anovulation and, in some cases, to resistance to clomiphene citrate therapy.

Bacteremia in post-Cesarean section endomyometritis: differential response to therapy.

Presented are blood culture results obtained from 200 patients with post-cesarean section endomyometritis treated with either penicillin-gentamicin or clindamycin-gentamicin. Their clinical course is correlated to their blood culture results by the fever index. Fifty-three percent of the 60 organisms isolated from 48 patients were anaerobic bacteria. Patients from whose blood cultures anaerobic bacteria were recovered had higher fever indexes than did those with aerobic isolates (P less than .05). Clindamycin-gentamicin patients from whose blood cultures anaerobic organisms were isolated had less febrile morbidity than did comparable penicillin-gentamicin patients. Patients with Bacteroides fragilis bacteremia had the highest fever indexes overall. Therefore, patients with post-cesarean section endomyometritis have less febrile morbidity if they are initially treated with a drug effective against anaerobic bacteria, especially B fragilis.

Reduction of retrosternal and pericardial adhesions with rapidly resorbable polymer films.

BACKGROUND: The formation of postoperative cardiac adhesions makes a repeat sternotomy time consuming and dangerous. Many attempts have been made to solve this problem by using either drugs to inhibit fibrinolytic activity or different types of pericardial substitutes. The results have not been satisfactory. METHODS: The efficacy of bioresorbable film prototypes made of polyethylene glycol (EO) and polylactic acid (LA) (EO/LA = 1.5, 2.5, and 3.0) in the prevention of adhesions after cardiac operations in canine models was tested. After desiccation and abrasion of the epicardium, a transparent bioresorbable film was placed over the heart. The pericardium was closed to allow intrapericardial adhesions (n = 32) or left open and attached to the chest wall to induce retrosternal adhesions (n = 17). Postoperative recovery was similar among the groups. Retrosternal and pericardial adhesions were evaluated at necropsy 3 weeks later by assessing area, tenacity, and density of the adhesions. RESULTS: In the control dogs, tenacious, dense adhesions were observed. In contrast, adhesion formation was reduced at all sites covered by the films. The bioresorbable films were efficacious in the reduction of adhesion formation between epicardium and pericardium or between epicardium and sternum after cardiac operation. The EO/LA 1.5 film most effectively prevented the early adhesions. CONCLUSIONS: The bioresorbable films (EO/LA = 1.5, 2.5, and 3.0) significantly reduced adhesion formation, with EO/LA = 1.5 (Repel CV) being optimal. As the barrier was rapidly resorbed, the capsule formation induced by permanent barriers was avoided.