Comparison of controlled ovarian stimulation with human menopausal gonadotropin or recombinant folliclestimulating

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1 FERTILITY AND STERILITY VOL. 80, NO. 2, AUGUST 2003 Copyright 2003 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. Comparison of controlled ovarian stimulation with human menopausal gonadotropin or recombinant folliclestimulating hormone Marco Filicori, M.D., Graciela E. Cognigni, M.D., Patrizia Pocognoli, M.D., Cristina Tabarelli, M.D., Federica Ferlini, M.D., Tiziana Perri, M.D., and Lodovico Parmegiani, B.Sc. Reproductive Endocrinology Center, University of Bologna, Bologna, Italy Received October 28, 2002; revised and accepted January 16, Dr. Marco Filicori is a scientific advisor to Ferring S.P.A. Reprints requests: Marco Filicori, M.D., Reproductive Endocrinology Center, Department of Obstetrics and Gynecology, University of Bologna, Via Massarenti 13, Bologna, Italy (FAX: ; marco.filicori@ unibo.it) /03/$30.00 doi: /s (03) Objective: To carefully examine the features of controlled ovarian stimulation performed with recombinant FSH- or hmg. Design: Controlled, prospective, randomized comparison of fixed gonadotropin regimens. Setting: Academic research institution. Patient(s): Fifty infertile patients who were candidates for IUI. Intervention(s): Patients were randomized to receive a fixed regimen of recombinant FSH- (150 IU/day, 25 patients) or hmg (150 IU/day, 25 patients), after GnRH-agonist suppression (long regimen). Main Outcome Measure(s): Daily measurements of serum LH, immunoreactive FSH, hcg, E 2, P, and T. Transvaginal pelvic ultrasound every 2 days. Pregnancy and abortion rates. Cost of medications. Result(s): Two recombinant FSH- -treated patients did not respond. Despite matched daily FSH dose, duration of treatment (hmg vs. recombinant FSH days), gonadotropin dose ( vs ampoules), gonadotropin cost ( vs. 1, /cycle), serum P levels, and small preovulatory follicle number were significantly lower, and LH, hcg, immunoreactive FSH levels, and larger follicles on day 8 were significantly higher in hmg-treated patients. The pregnancy, abortion, and twin pregnancy rates did not differ. Conclusion(s): The hmg administration was associated with: [1] increased serum LH activity and immunoreactive FSH levels during treatment; [2] reduced signs of premature luteinization; [3] differential modulation of folliculogenesis; [4] lower treatment duration, gonadotropin dose, and cost; and [5] clinical outcome comparable to recombinant FSH-. (Fertil Steril 2003;80: by American Society for Reproductive Medicine.) Key Words: Ovulation induction, luteinizing hormone, recombinant follicle-stimulating hormone, human menopausal gonadotropin, ovarian follicle, intrauterine insemination Intrauterine insemination (IUI) preceded by controlled ovarian stimulation (COS) has become a common therapeutic procedure among infertile couples. Exogenous gonadotropins are used to optimize folliculogenesis and to provide higher chances of fertilization through a more precise timing for IUI and the availability of greater oocyte number, particularly when the sperm count is reduced. Several drugs with different hormone contents and characteristics are available from pharmaceutical companies for this type of treatment. Recombinant FSH is devoid of LH activity, whereas hmg is known to contain measurable amounts of both LH and hcg. Although some pharmaceutical companies are known to add hcg to hmg to provide adequate amounts of LH activity (so-called spiking), unintended contamination from urines of pregnant donors and other sources may occur (1). In addition, the properties of FSH present in these preparations appear to differ: human-derived FSH contains more acidic FSH isoforms that are associated with a longer serum half-life (2), whereas the less acidic FSH isoforms present in recombinant FSH have a shorter half-life but stimulate estrogen (E) secretion more efficiently (3). 390

2 Despite these relevant differences in gonadotropin type, content, and activity, and their widespread use, few studies have carefully compared treatment characteristics and outcome in patients treated with different medications. In particular, no study has so far prospectively compared recombinant FSH and hmg in an IUI program. Although clinical efficacy of COS is the foremost concern of clinicians in this area, greater understanding of the specific effects and mechanisms of action of each gonadotropin preparation is critical to optimize and improve treatment. To achieve this goal, controlled drug administration and careful monitoring are essential to ensure that each medication is properly tested. Thus, we elected to compare the administration of fixed amounts of recombinant FSH- and hmg in patients who were candidates for IUI and to assess treatment with daily blood samples for hormone determinations and pelvic ultrasound performed at alternate days. All patients concomitantly received a depot GnRH-agonist (GnRH-a) administered in the long regimen to ensure that the contribution of endogenous gonadotropins was limited. Recombinant FSH is currently considered by many investigators as superior to human-derived gonadotropins (4, 5). For this reason we assigned patients treated with recombinant FSH- to our reference group (A) and compared them to patients treated with the same daily amounts of hmg (group B). The results of this study, however, did not confirm this purported superiority of recombinant FSH- and provided novel and valuable information on the endocrine, folliculogenetic, and clinical characteristics of gonadotropin treatment that may permit optimization and reduction of the cost of COS in infertile patients. MATERIALS AND METHODS Patient Population A total of 50 patients, aged years, and diagnosed as having unexplained or mild male factor-related infertility were studied. All subjects had regular menstrual cycles of 25- to 32-day duration, a body mass index (BMI) of kg/m 2, a hysterosalpingogram or laparoscopy demonstrating tubal patency, a pelvic ultrasound showing a uterus and ovaries of normal size and structure (no signs of polycystic ovary syndrome [PCOS]), normal baseline biochemical and endocrine determinations (including thyroid hormones), reproductive hormones within the normal range for the early/ midfollicular phase of the cycle, and no history or signs of endometriosis. Although ovulation induction had been previously performed in some of the subjects (9 in group A and 13 in group B, P not significant [NS]), no patient had received any hormone therapy (including gonadotropins) for at least 3 months preceding the study. Protocol Our Institutional Review Board approved the protocol and all patients provided informed consent. Patients were then randomly assigned to two age- and weight-matched groups: group A that received recombinant FSH- (Gonal-F, Serono Pharma S.p.A., Rome, Italy; cost per 75 IU ampoule 51.39) and group B that received hmg (Menogon, Ferring S.p.A., Milan, Italy; cost per 75 IU ampoule 13.28). Patients were not blinded to treatment, which was started in the midluteal phase of a spontaneous menstrual cycle with the administration of a single injection of 3.75 mg of depot Triptorelin (Decapeptyl 3.75, IPSEN S.p.A., Milan, Italy). Controlled ovarian stimulation began 14 days thereafter. All patients menstruated before the initiation of gonadotropin treatment and pelvic ultrasound showed no formation of ovarian cysts after GnRH-a administration. The gonadotropin regimens adopted in each group were for group A recombinant FSH- 150 IU, SC, daily, and for group B hmg 150 IU, IM, daily. This gonadotropin dose was continued until the occurrence of at least two ovarian follicles 17 mm in diameter and of serum E 2 concentrations 600 pg/ml (final maturation parameters) or for 14 days if the final maturation parameters were not achieved. After the 14th day of treatment, increments of the gonadotropin dose were allowed (225 IU/day on days and 300 IU/day on days 18 20). Treatment was discontinued if the patient menstruated during treatment or if the final maturation parameters were not reached by day 21. In all patients gonadotropins were administered between 4 and 6 P.M. When the final maturation parameters were attained, 10,000 IU of hcg (Profasi, Serono Pharma S.p.A.) was administered to trigger final follicular maturation and IUI with a sperm swim-up procedure was performed 36 hours thereafter. The luteal phase was supported with 90 mg daily of intravaginal P gel (Crinone, Serono Pharma S.p.A.) administered from the third to the 14th day after the preovulatory hcg dose. Monitoring Treatment monitoring was conducted throughout gonadotropin administration. Each day one blood sample was drawn between 8 and 9 A.M. in a standard manner, and two serum aliquots were obtained: E 2 was measured daily in one of the serum aliquots for clinical monitoring and the second aliquot was stored at 20 C for later measurements of LH, FSH, hcg, E 2, P, and T. Transvaginal pelvic ultrasound was performed during gonadotropin treatment on days 0 and 6 and at alternate days thereafter, until preovulatory hcg administration or treatment discontinuation. Ovarian follicles detected at each ultrasound examination were classified as small ( 10 mm diameter), intermediate (10 14 mm), and large ( 14 mm). The physician performing pelvic ultrasound was blinded as to which arm of the protocol each patient belonged. FERTILITY & STERILITY 391

3 Hormone Assays Luteinizing hormone, FSH, hcg, E 2, P, and T were measured with chemiluminescence assays (Chiron Diagnostics ACS 180, Milan, Italy). The minimal detectable dose (MDD) of LH was 0.1 IU/L. The interassay CVs at low, intermediate, and high levels of the standard curve were 4.0%, 5.2%, and 3.9%, respectively. The in vitro addition of up to 200,000 IU/L of hcg did not affect LH determinations in this assay, as assessed at multiple levels of the standard curve. The MDD of FSH was 0.3 IU/L. The interassay CVs at low, intermediate, and high levels of the standard curve were 2.4%, 5.7%, and 4.0%, respectively. The MDD of hcg in this -specific assay was 0.1 IU/L. The interassay CVs at low, intermediate, and high levels of the standard curve were 4.3%, 3.8%, and 3.6%, respectively. The in vitro addition of up to 200 IU/L of LH did not affect hcg determinations in this assay, as assessed at multiple levels of the standard curve. TheMDDofE 2 was 10 pg/ml. The interassay CVs at low, intermediate, and high levels of the standard curve were 6.2%, 4.5%, and 4.9%, respectively. The MDD of P was 0.1 ng/ml. The interassay CVs at low, intermediate, and high levels of the standard curve were 3.9%, 2.0%, and 4.5%, respectively. The MDD of T was 0.1 ng/ml. The interassay CVs at low, intermediate, and high levels of the standard curve were 5.6%, 1.4%, and 4.0%, respectively. Statistical Evaluation Data were expressed as mean SE. Serum hormone levels were calculated in each cycle as area under the curve (AUC). Normality of distribution of continuous variables was assessed with a Kolmogorov-Smirnov test (with Lilliefors correction). Between-group differences of normally distributed continuous variables were assessed with parametric statistics (Student s t test), whereas nonparametric statistics (Mann-Whitney rank sum test) were used when the normality test was not passed. Between-group differences in noncontinuous variables were assessed with the 2 method with the Yates correction, if needed. RESULTS The baseline patient characteristics of the two treatment groups are shown in Table 1. No significant between-group differences existed in these parameters, including age, BMI, ovarian volume, reproductive hormone levels, and partner s sperm quality. All the patients of group B completed treatment and received preovulatory hcg; conversely, two patients of group A failed to achieve the protocol s final maturation parameters ( 2 follicles 17 mm, and serum E pg/ml) by treatment day 21 and thus treatment was discontinued before hcg administration (P NS). Eleven ultrasound-detected pregnancies were obtained, four in group A and seven in group B, corresponding to TABLE 1 Baseline characteristics of the patients randomized to be treated with recombinant FSH- or hmg. Baseline parameters Group A (recombinant FSH- ) Group B (hmg) Number of patients Age (years) Height (cm) Weight (kg) BMI (kg/m 2 ) Menstrual cycle duration (days) Mean ovarian volume (cm 3 ) Partner s sperm count (millions/ml) Partner s sperm motility (% at 120 min) LH (IU/L) FSH (IU/L) PRL (ng/ml) E 2 (pg/ml) P (ng/ml) T (ng/ml) Note: P value is not significant. pregnancy rates per completed cycles of 17% and 28%, respectively (P NS). There were five twin pregnancies, two in group A and three in group B (P NS), and one spontaneous abortion in each group (P NS). None of the treatment cycles resulted in moderate or severe ovarian hyperstimulation syndrome (OHSS). The results of therapy were analyzed using an intent to treat paradigm, that is, outcomes assessment (except for the preovulatory E 2 concentrations and preovulatory follicle pattern) included all patients, regardless of their achievement of the final maturation parameters. Treatment duration (group A vs. group B days/cycle, P.05), the gonadotropin dose used (group A vs. group B ampoules/cycle, P.05), and the gonadotropin cost (group A 1, vs. group B /cycle, P.001; Fig. 1) were significantly reduced in the patients treated with hmg (Table 2). The AUC of reproductive hormones are shown in Table 2. The AUC of serum LH concentrations (group A vs. group B IU/L.day, P.05; Fig. 2), immunoreactive FSH (group A vs. group B IU/L daily, P.01; Fig. 3), and hcg (group A 0.1 vs. group B IU/L daily, P.01; Fig. 4) were increased in the patients receiving hmg. Conversely, serum P concentrations (group A vs. group B ng/ml daily, P.001; Fig. 5) were increased in the patients receiving recombinant FSH-. Serum E 2 (group A 1, vs. group B 2, pg/ml daily, P NS), and T levels (group A vs. group B ng/ml.daily, P NS) did not differ. 392 Filicori et al. Features of hmg vs. recombinant FSH Vol. 80, No. 2, August 2003

4 FIGURE 1 Gonadotropin cost per treatment cycle needed to achieve comparable levels of folliculogenesis in patients treated with recombinant FSH- or hmg. Values are shown as mean SE. *P.01. Preovulatory E 2 concentrations were not different in the two treatment groups (group A 1, pg/ml vs. group B 1, pg/ml, P NS; Table 2). The development of large ovarian follicles was hastened in patients receiving hmg, as the number of follicles 14 mm was significantly increased on treatment day 8 in the patients of group B (group A vs. group B 1.9 TABLE 2 Features of treatment in patients receiving recombinant FSH- or hmg. Variables Group A (recombinant FSH- ) Group B (hmg) P value Treatment characteristics Duration of COS (days/cycle) Gonadotropin dose (ampules/cycle) Reproductive hormones across treatment (AUC) LH (IU/L.day) FSH (IU/L.day) hcg (IU/L.day) E 2 (pg/ml.day) 1, , NS P (ng/ml.day) T (ng/ml.day) NS Day of hcg administration E 2 (pg/ml) 1, , NS Ovarian follicles 10 mm mm NS 14 mm NS Treatment outcome Pregnancies (rate) 4 (17%) 7 (28%) NS NS not significant. 0.7, P.05). However, in the last pelvic ultrasound performed before hcg administration (Table 2), the number of large (group A vs. group B , P NS) and intermediate (group A vs. group B , P NS) preovulatory follicles did not differ, whereas the number of small preovulatory follicles was reduced in patients receiving hmg (group A vs. group B , P.001). DISCUSSION Few investigations have prospectively compared recombinant FSH to human-derived gonadotropin preparations containing both FSH and LH activity such as hmg or highly purified hmg (6 9). Thus, we elected to conduct a detailed and carefully controlled study to clarify the differences existing in ovarian folliculogenesis, serum hormone levels, and treatment characteristics in two groups of patients receiving recombinant FSH- or hmg. The key features of our protocol, rarely found in other studies, were the use of fixed and equal amounts of FSH (150 IU/day for up to 14 days), regardless of the patients response, the administration of a potent depot GnRH-a given in the long regimen, and the insistence on minimal levels of both follicle development and serum E 2 concentrations (final maturation parameters) before triggering ovulation with hcg. This experimental design ensured consistency in the assessment of the different stimulatory regimens, negligible interference from endogenous gonadotropin secretion, and a limited impact of confounding variables such as drug dose upon treatment outcome. As expected from previous studies from our (10) and other groups (11), serum levels of LH (Fig. 2) and hcg (Fig. 4) were higher in hmg- than in FSH-treated patients. This finding confirmed that the higher LH activity present across the follicular phase of hmg cycles is related to increments of both LH and hcg levels (1). Additional LH activity may result from the contribution of residual endogenous LH secretion, particularly when less potent GnRH agonists types or regimens are used. This activity and the use of heterogeneous gonadotropin dosages may explain the lack of differences in the stimulation outcome parameters encountered in some previous comparisons of FSH regimens with and without LH activity supplementation (7, 12). A somewhat unexpected feature of this study was the finding of significantly lower levels of immunoreactive FSH in patients treated with recombinant FSH- (Fig. 3). The different routes of gonadotropin administration used in this study (SC for recombinant FSH, IM for hmg) are unlikely to have caused this difference as daily SC administration of 150 IU of recombinant FSH resulted in higher peripheral serum FSH levels than 150 IU of recombinant FSH given IM (13), and FSH bioequivalence was found after IM and SC hmg administration (14). Conversely, recombinant FSH preparations are known to contain higher amounts of less acidic FERTILITY & STERILITY 393

5 FIGURE 2 Daily serum LH concentrations (left) and AUC (right) during the administration of 150 IU/day of recombinant FSH- (group A) or 150 IU/day of hmg (group B). Values are shown as mean SE. FSH isoforms that are associated with a shorter plasma half-life than the more acidic FSH isoforms present in human-derived FSH (15). Thus, the most likely explanation for the findings of our study is that this isoform profile negatively affected immunoreactive FSH concentrations in patients treated with recombinant FSH-. Assessments of immunoreactive FSH pharmacokinetics after single bolus administration showed higher serum FSH levels after human-derived highly purified FSH than after recombinant FSH (2). However, the results of our study for the first time indicated that differences in immunoreactive FSH levels were also present after hmg administration and that this profile persisted over an entire COS cycle. Additional supporting evidence for the findings of the present study derive from a recently reported investigation where a similar difference in immunoreactive serum FSH was found when recombinant FSH- and highly purified hmg were compared (9). The faster clearance of immunoreactive FSH FIGURE 3 Daily serum FSH concentrations (left) and AUC (right) during the administration of 150 IU/day of recombinant FSH- (group A) or 150 IU/day of hmg (group B). Values are shown as mean SE. 394 Filicori et al. Features of hmg vs. recombinant FSH Vol. 80, No. 2, August 2003

6 FIGURE 4 Daily serum hcg concentrations (left) and AUC (right) during the administration of 150 IU/day of recombinant FSH- (group A) or 150 IU/day of hmg (group B). Values are shown as mean SE. from the peripheral circulation during recombinant FSH- administration may result in a more rapid decrement or cessation of ovarian stimulation when this medication is reduced or discontinued. Thus, the findings of our study may have further clinically relevant implications in conditions such as coasting (16) when an excessively abrupt cessation of FSH stimulation may hamper follicle survival. The increased content of less acidic FSH isoforms typical of recombinant FSH preparations appears to be also associated with greater potency of these compounds in their capacity to stimulate steroid biosynthesis from granulosa cells (3). This feature can explain our finding of higher serum P during recombinant FSH- treatment (Fig. 5), and of no statistically significant differences in serum E 2 levels between groups A and B (Table 2). In a previous similarly conducted study (10) where we compared hmg to humanderived highly purified FSH, P levels did not differ and E 2 secretion was significantly increased in the hmg-treated FIGURE 5 Daily serum P concentrations (left) and AUC (right) during the administration of 150 IU/day of recombinant FSH- (group A) or 150 IU/day of hmg (group B). Values are shown as mean SE. FERTILITY & STERILITY 395

7 patients. Thus, in the current study the greater biopotency of recombinant FSH to stimulate steroidogenesis could have compensated for the reduced serum immunoactive FSH levels we encountered in recombinant FSH- -treated patients. The presence of higher serum P levels in group A (Table 2) confirmed that moderate follicular phase serum P increments are related to FSH and not to LH activity administration (1, 17, 18), and that so-called premature luteinization is directly linked to FSH granulosa cell stimulation. This interpretation is further supported by our finding of increased P serum levels even in the early stages of COS (Fig. 5), that is, when granulosa cells do not yet express LH receptors and are thus insensitive to LH stimulation. Finally, the finding of low serum T levels during COS in both treatment groups (Table 2) confirmed that the administration of standard hmg dosages, albeit capable of stimulating T secretion in a dosedependent manner (19), will not result in excessive serum T levels during COS (10, 20). Our finding of comparable levels of intermediate and large preovulatory follicles (Table 2) confirmed that both hmg and recombinant FSH- can generally stimulate folliculogenesis to a similar degree. Nevertheless, various features differed between groups A and B. Large follicles ( 14 mm) emerged sooner (day 8 of stimulation), and the incidence of small preovulatory follicles ( 10 mm) was significantly reduced in hmg-treated patients (Table 2). These traits indicated that LH activity is capable of directly modulating folliculogenesis, albeit in a different manner from FSH (1). The stimulatory effect of LH activity on larger follicles is the likely consequence of LH receptors expression on the granulosa cells of these follicles (21 24), whereas small follicle demise may be mediated by increased intrafollicular androgen concentrations (25). We recently demonstrated that the action of LH activity on small follicles is dose dependent (19), and that it is mirrored by changes in serum inhibin concentrations (10). As the occurrence of fewer small preovulatory follicles appears to be positively correlated with a reduced chance of developing OHSS (26), gonadotropin regimens that provide higher LH-to-FSH ratios in the last COS stages (27, 28) may cause fewer ovulation induction complications. Treatment outcome in terms of pregnancy and complication rates was comparable in groups A and B. Nevertheless, the use of hmg was associated with a reduced duration of administration and dosage of gonadotropins, and lower drug cost (Fig. 1). In a previous study where we compared highly purified FSH to hmg (both human-derived drugs) we found a similar improvement in treatment profile associated with hmg (10). Thus, our interpretation of the results of the current study is that our findings are likely related to the added stimulatory activity of LH activity on the granulosa cells of larger ovarian follicles (1), albeit an additional effect of the higher serum immunoreactive FSH found in hmgtreated patients cannot be ruled out. In summary, this study confirmed our previous findings on the action of LH activity in COS (10, 19), and extended them to recombinant FSH preparations. Additional investigations will be needed to further characterize the features and specific merits of treatment conducted with recombinant and human-derived gonadotropins, and to better assess the potentials of regimens that combine FSH and LH activity administration in traditional and unconventional therapies (29). 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