The need to step up the gonadotropin dosage in the stimulation phase of IVF treatment predicts a poor outcome.

PURPOSE: It is a common practice to increase the gonadotropin dose during ovarian stimulation when the estradiol (E2) rise is found to be inadequate. The prognostic impact of the use of this step-up regimen on the outcome of the affected in vitro fertilization (IVF) cycle is the subject of this study. METHODS: This is a retrospective analysis of IVF cycles in a series of consecutive patients who required an increase in the gonadotropin dosage during the stimulation phase because of inadequate E2 rise. Controls consisted of patients in whom the dose was not increased. After 4 days of stimulation, the gonadotropin dosage was increased if E2 levels failed to rise by 70% every 2 days. Outcome was defined in terms of maximum E2 level, number of follicles at aspiration, number of oocytes obtained, fertility rate, and pregnancy rate and was compared in study and control patients. Pregnancy was defined by sonographic demonstration of cardiovascular activity. RESULTS: One hundred forty-five patients were analyzed. A step-up regimen was used in 35 patients (24.1%). Patients who required the step-up dosing had significantly lower peak E2 levels (1373 vs 1828 pg/ml; P < 0.005), fewer follicles measuring greater than 16 mm (7.2 vs 9.7; P < 0.003), and fewer oocytes recovered (8.3 vs 11.2; P < 0.009). The fertilization rate (67.6 vs 64.2%) was not significantly different. The pregnancy rate (8.5 vs 32.7%; P < 0.004) was significantly lower in the group requiring the step-up regimen. CONCLUSIONS: The utilization of a step-up regimen during an IVF treatment cycle is a predictor of a poor outcome for the specific IVF cycle. As this information is available before retrieval, consideration of cycle cancellation may be appropriate.

Human follicular fluid contains a follicle-stimulating hormone (FSH) receptor binding inhibitor which has FSH agonist activity, is immunologically similar to FSH, but can be distinguished from FSH.

We have previously reported the presence in human follicular fluid (hFF) of a high (greater than 5000) mol wt FSH receptor binding inhibitor (FSH-BI). This hFF FSH-BI was further purified by removal of material insoluble in acidified acetone (pH 4.1) but soluble in diethyl ether (pH 10.5), followed by molecular sieving through Sephacryl S-100. FSH-BI activity eluted from S-100 with an elution volume similar to that of hFSH, but could be distinguished from hFSH on the basis of a differential sensitivity to acid inactivation. Human FSH was inactivated in acetone at pH 4.1 (1 h, 25 C), whereas hFF FSH-BI retained activity under these conditions. Human FF FSH-BI also demonstrated FSH-like agonist activity, defined as the ability to stimulate basal levels of estradiol synthesis in cultured rat Sertoli cells. Human FSH-BI strongly cross-reacted to a commercially available monoclonal antibody used to measure serum hFSH. Indeed, recovery of FSH immunologic activity was significantly greater (134-fold on a mass basis) after partial purification, indicating that antibody recognition sites were apparently masked in unfractionated hFF. In summary, large mol wt hFSH-BI has been partially purified from hFF and found to be similar in size to pituitary hFSH and to have FSH-like agonist activity in vitro. Although distinguishable from pituitary hFSH on the basis of stability to acid, hFSH-BI appears immunologically related to pituitary hFSH so that measurements of hFSH levels in hFF using immunologic techniques should be interpreted with caution.

Identification of low and high molecular weight follicle-stimulating hormone receptor-binding inhibitors in human follicular fluid.

Various fractions of human follicular fluid (FF) were tested for follicle-stimulating hormone (FSH) receptor-binding inhibitory activity, as well as for their ability to form a dansyl derivative previously reported to be associated with FSH receptor-binding inhibitory activity in bovine FF. Pooled human FF was found to inhibit binding of radioiodinated human FSH (125I-hFSH) to its receptor in a concentration-related manner. Fractionation of human FF by sequential ultrafiltration through membranes of calibrated pore size resulted in human FF components of low (500 to 5,000) and high (greater than 5,000) molecular weight, (Mr) respectively. Each of these inhibited 125I-hFSH binding to receptor in a concentration-related manner. Dansylation of the above components revealed the Rf = 0.15 dansyl derivative to be present in unfractionated human FF and in the low (500 to 5,000 Mr), but not the high (greater than 5,000 Mr), component of human FF. These findings are consistent with those previously reported for bovine FF and porcine FF and indicate that human FF contains FSH receptor-binding inhibitors with properties similar to those observed in animal species.

The relationship of sperm parameters to cycle fecundity in superovulated women undergoing intrauterine insemination.

Semen parameters of raw and prepared (post-swim-up) specimens from 451 cycles of intrauterine insemination (IUI) were analyzed in relation to cycle fecundity in 232 patients undergoing ovarian stimulation with sequential clomiphene citrate/menotropin therapy. Pregnancy occurred in 42 cycles, resulting in an overall pregnancy rate of 17.7%, and a cycle fecundity of 9.3%. Cycle fecundity was positively correlated with the parameters of post-swim-up log sperm density (r = 0.994), and with log total motile sperm inseminated (r = 0.964; inseminates were limited to a maximum of 20 million total motile sperm). Post-swim-up motility did not correlate (r = 0.308) with cycle fecundity; however, most specimens had a motility of greater than 40% post-swim-up. Only one pregnancy occurred when less than 1 million motile sperm were inseminated (38 cycles), which resulted in a cycle fecundity of 2.6% for these cycles. This may represent the threshold of effectiveness for IUI in this setting. Highest cycle fecundity was obtained with an inseminate containing approximately 10 million or more motile sperm. Parameters of raw samples correlated less well with cycle fecundity than did prepared specimens. Analysis of post-swim-up semen parameters can provide useful prognostic information for women undergoing IUI with ovarian stimulation; this information is helpful in counseling patients regarding their chances of success with this therapy.

A prospective study on the lack of development of antisperm antibodies in women undergoing intrauterine insemination.

To test the hypothesis that intrauterine insemination with washed spermatozoa induces antisperm antibody formation, we measured serum antisperm antibody levels by the Immunobead technique in a population of women receiving exogenous gonadotropins. Antibody levels were measured before therapy (baseline) and then serially during subsequent stimulation cycles, for a maximum of six cycles. Twenty-eight patients underwent intrauterine insemination; each patient served as her own control. An additional 25 patients were treated with exogenous gonadotropins but did not undergo intrauterine insemination; they served as external controls. Antisperm antibody levels in women who underwent concomitant intrauterine insemination were compared with levels in those who did not. Of the 53 enrolled patients, 18 completed six treatment cycles, and 35 achieved pregnancy before six cycles. Forty-five patients (85%) had less than 10% Immunobead binding, six (11%) had binding between 10% and 25% (mean 16%, range 14% to 20%), and two had binding greater than 25% (28% and 42%, respectively). Mean binding was similar (less than 10%) in the intrauterine insemination and external control groups. Eighteen patients conceived in the intrauterine insemination group and seventeen in the control group. Of patients who conceived, all but one had less than 10% Immunobead binding at the time of conception (mean 1.6 months). In patients who did not conceive, there was no difference in Immunobead binding between control and intrauterine insemination groups after 6 months of therapy. Our data do not support the hypothesis that serum antisperm antibody levels, as detected by Immunobead binding, will increase in menotropin-stimulated women undergoing intrauterine insemination over a prolonged treatment period.

Exogenous gonadotropin requirements are increased in leuprolide suppressed women undergoing ovarian stimulation.

We used a subcutaneously administered GnRHa for 21 to 26 days prior to menotropin stimulation, to suppress endogenous LH surges in four patients participating in IVF. GnRHa-pretreated cycles were compared with previous menotropin treatment cycles. Endogenous LH surges were successfully suppressed in all patients. Peak E2 levels and ultrasonographic parameters of follicular development were comparable in the two treatment groups. Exogenous gonadotropin requirements were increased 2- to 4-fold in GnRHa pretreated cycles (P less than 0.05). Ovum recovery rates were not improved by adjuvant LA. These studies indicate that there was an increased ovarian requirement for exogenous gonadotropins as a result of GnRHa therapy. It has to be considered that this may be a direct effect of GnRHa upon the ovary. Alternatively, the absence of endogenous pituitary support in GnRHa-treated patients may account for the increased gonadotropin requirement. Finally, it is possible that this effect is indigenous to this select patient population of poor responders to menotropin stimulation, or to a specific effect of subcutaneously (as opposed to intranasally) administered GnRHa on the ovary. Further studies are needed to clarify the extent to which any or all of these postulated mechanisms may be influencing ovarian response to exogenous gonadotropins after subcutaneous GnRHa pretreatment.

Pixe analysis of reproductive fluids.

An external 3.8-MeV proton beam was employed to induce X-rays in 100-mg pellets of human follicular fluids and in 4-8 mg pellets of spermatozoa. The elements Cl, K, Ca, Fe, Cu, Zn, and Br were quantitatively determined in follicular fluids, whereas the elements, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Ni, Cu, and Zn were determined in the spermatozoa. Both samples had high interelement Spearman correlation coefficients. Correlations of elements in the samples were observed with several clinical parameters. The multielemental analysis of spermatozoa can be used to approximate quantities of trace elements inserted into the egg ooplasm at the time of fertilization. These elemental quantities appear to be in the femtogram (10(-15)) range.

Use of antisera to follicle-stimulating hormone (FSH) to detect non-FSH factors in human serum which modulate rat granulosa cell steroidogenesis.

We measured the ability of serum from women to stimulate steroidogenesis in cultured granulosa cells. Serum promoted estradiol and progesterone synthesis in proportion to its FHS content measured by RIA [i.e. serum from postmenopausal women (PM) greater than serum from the midcycle at the time of the gonadotropin surge (MC) greater than serum from the first day of the menstrual cycle (D1) greater than serum from a hypophysectomized woman (AP)]. The FSH activity of these sera was reduced but not eliminated when we included excess antisera to ovine or human FSH in the culture medium (i.e. PM greater than MC greater than D1 greater than AP). These antisera completely neutralized the actions of ovine FSH, human FSH, and menopausal gonadotropin (Pergonal) added to serum. In contrast to the stimulation seen with 5% or lower concentrations of serum in the culture medium, we observed that 10-20% serum inhibited FSH-induced androgen aromatization and progesterone accumulation. The degree of stimulation or inhibition of steroidogenesis depended on the number of granulosa cells added to each culture. High initial cell concentrations inhibited the ability of the cells to respond to either serum or PMSG. In addition to factors which stimulate or inhibit FSH-induced steroidogenesis, human serum contains factors distinct from FSH which cause the cells to flatten and adhere more tightly to the culture dishes. Although progesterone synthesis was increased in cells which had flattened on the surface of the culture dishes, this phenomenon was not a prerequisite for serum-induced steroidogenesis. We conclude that serum contains factors immunologically distinct from FSH, possibly of pituitary origin, which induce granulosa cell steroidogenesis. In addition, serum contains inhibitory substances which block hormone-induced steroidogenesis and which tend to obscure the stimulatory effects of FSH. Detection of both factors depends in part on the number of granulosa cells used to innoculate the cell cultures.

The initial experience with the use of a portable infusion pump in the delivery of human menopausal gonadotropins.

The initial experience with the use of a portable infusion pump for the delivery of human menopausal gonadotropins (hMG) in the therapy of ovulation dysfunction of women who failed to conceive following (1) either clomiphene citrate (five patients with polycystic ovary syndrome) or danazol (three patients with mild endometriosis) and (2) standard intramuscular hMG-human chorionic gonadotropin therapy is reported. In six of these patients this approach was used because of suboptimal response of serum estradiol levels to standard hMG therapy, and all six patients had an increase in estradiol response with infusion therapy; therapy duration and total dosage of hMG were similar in both treatment modalities. Sonography suggested stimulation of several follicles with infusion therapy. Advantages and disadvantages of the use of the infusion pump for hMG therapy are discussed. In conclusion, hMG therapy via the infusion pump is feasible. This mode of administration appears to lead to more satisfactory follicular development in selected patients. Multiple follicular stimulation is to be expected. Self-administration of medication is readily achieved by the educated patient. Further consideration and exploration of hMG infusion therapy is suggested.

Induction of ovulation with menotropins in women with polycystic ovary syndrome.

Twenty-four women with ovulatory infertility as a result of surgically or biochemically documented polycystic ovary syndrome (PCO) who had failed to conceive during clomiphene citrate therapy underwent a closely supervised menotropin treatment to induce ovulation. Evidence of ovulation was obtained in all patients treated, and major side effects were limited. Fourteen women conceived after an average of 2.4 treatment cycles; twin pregnancies occurred in 36% and spontaneous abortions occurred in 21%. Initial treatment cycles tended to be less successful than the subsequent treatment cycles. Serum 17 beta-estradiol (E2) levels were significantly augmented in the last 3 days before administration of chorionic gonadotropins (hCG) in treatment cycles resulting in conception compared to E2 levels in those cycles which resulted in ovulation only. A second hCG administration to trigger ovulation had to be given in 27% of the treatment cycles and seemed to be an indication of a less promising treatment cycle. Treatment cycles resulting in twin gestations did not differ from those resulting in singleton gestations; specifically, the E2 response was not increased. In summary, under a closely monitored regimen, menotropin therapy can be used in women with nonovulatory infertility as a result of PCO with considerable effectiveness and relative safety once clomiphene citrate treatment has failed.

Response of blood coagulation parameters to elevated endogenous 17 beta-estradiol levels induced by human menopausal gonadotropins.

To elucidate the relationship between estrogen and thrombosis, we studied blood coagulation parameters in women whose ovaries were stimulated with human menopausal gonadotropins (hMG). Daily hMG administration over 1 to 2 weeks in seven anovulatory women increased plasma 17 beta-estradiol levels fivefold over the pretreatment value. Of the coagulation parameters, the fibrinogen level increased significantly from an initial value of 248 +/- 11.7 mg/dl (mean +/- SEM) to 353 +/- 32.2 mg/dl after hMG treatment (P less than 0.05), with a significant positive correlation between estrogen and fibrinogen levels (r = +0.762). In addition, a thrombokinetics study showed that the maximal rate of change in optical density of the prothrombin time and activated partial thromboplastin time was significantly increased, suggesting that the coagulation factors involved in extrinsic, intrinsic, and common pathways could be increased by estrogen. Antithrombin III levels decreased gradually during hMG administration. Thus, increased endogenous estrogen levels appear to induce the so-called "hypercoagulable state" through both an increase in coagulation factors in the coagulation cascade system and a decrease in antithrombin III, a potent natural inhibitor of activated coagulation factors. Patients on a regimen of hMG treatment for induction of ovulation serve as excellent models for the study of alteration of "natural" estrogen-mediated coagulation parameters.

Estrogenization in women with postmenopausal ovarian hyperthecosis.

The cases of two women who show postmenopausal bleeding and signs of endogenous estrogen production are presented. At laparotomy, ovarian hyperthecosis was found and confirmed histologically. Determination of 17 beta-estradiol concentrations in ovarian and peripheral veins suggested that these ovaries actively secreted excessive estrogens. Ovarian hyperthecosis is discussed as a cause of renewed endogenous estrogen activity in the postmenopausal women.

Effect of synthetic thyrotropin-releasing hormone on ovulation in baboons.

Twice daily oral thyrotropin-releasing hormone administered to female baboons throughout the menstrual cycle had no significant influence on cycle length or upon estrogen levels but produced blunted midcycle LH peaks and luteal phase progesterone levels in three fourths of the treatment cycles. Mean plasma prolactin levels were increased approximately 2.5-fold relative to untreated, ovulatory control cycles. CB-154 alone did not alter cycle length or endocrine parameters examined (mean prolactin levels were decreased but not significantly). Cycles during which CB-154 was administered concomitantly with TRH were characterized by normal ovulation as evidenced by luteal phase progesterone levels. Since the effect on LH secretion was reversed by concomitant CB-154 administration, TRH-induced anovulation in animals given long-term treatment appeared to be mediated through physiologic mechanisms sensitive to elevated circulating prolactin levels. However, this conclusion must be considered equivocal since prolactin levels were also elevated during ovulatory cycles following long-term TRH therapy. Finally, these data do no exclude the alternative possibility that anovulation in baboons given long-term TRH treatment was a reflection of thyroid disturbance and not directly attributable to elevated prolactin.