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Controlled ovarian stimulation with exclusive FSH followed by stimulation with hCG alone, FSH alone or hMG
European Journal of Obstetrics & Gynecology and Reproductive Biology 130 (2007) 99–106 www.elsevier.com/locate/ejogrb
Controlled ovarian stimulation with exclusive FSH followed by stimulation with hCG alone, FSH alone or hMG Mariana K.O. Gomes, Carolina S. Vieira, Marcos D. Moura, Luiz A. Manetta, Stael P. Leite, Rosana M. Reis, Rui A. Ferriani * Department of Gynaecology and Obstetrics of the University of Sa˜o Paulo Ribeira˜o Preto School of Medicine, Ribeira˜o Preto, Brazil Received 27 September 2005; received in revised form 16 April 2006; accepted 17 May 2006
Abstract Objective: To assess if low-dose hCG is similar to hMG and to rFSH in the late follicular phase. Study design: In a prospective randomized controlled trial, 51 patients undergoing controlled ovarian stimulation received ovarian priming with rFSH and then received hCG (200 IU/day) (hCG group, n = 17), hMG (225 IU/day) (hMG group, n = 17) or rFSH (200 IU/day) (FSH group, n = 17) in the late stage of follicular development. Parameters of follicular response and serum estradiol, progesterone and testosterone levels were assessed. Results: Pre-ovulatory ovarian follicle occurrence and length of treatment were similar among the three treatment groups. Serum progesterone level on the day of pre-ovulatory hCG was significantly higher in the hCG group than in the hMG or rFSH group. Clinical pregnancy rates were similar for all groups. The total cost of treatment was significantly lower for the hCG group than for the groups supplemented with hMG or rFSH. Conclusions: LH in the form of low-dose hCG during the late follicular phase induced the same follicular pattern as hMG and rFSH after ovulation induction. The procedure using hCG produced pregnancy rates similar to those obtained using hMG and rFSH, even though the patients showed higher serum progesterone levels on the hCG day. # 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Ovulation induction; Human chorionic gonadotropin; Human menopausal gonadotropin; Recombinant FSH; Luteinizing hormone
1. Introduction The importance of luteinizing hormone (LH) in the follicular phase and its role in ovulation induction are the subject of extensive debate. The optimal LH levels in assisted reproduction protocols and the drugs to be used for this purpose are still controversial topics [1–3]. Early follicular phase prevalence of follicle stimulating hormone (FSH) is responsible for follicle recruitment and the initial stimulation of the growth and development of granulosa cells in the pre-ovulatory follicle. In the middle * Corresponding author at: Av. Bandeirantes 3900, Campus Universita´rio, Departamento de Ginecologia e Obstetrı´cia da Faculdade de Medicina de Ribeira˜o Preto/USP, CEP 14049-900 Ribeira˜o Preto, SP Brasil. Tel.: +55 16 3602 2818; fax: +55 16 3633 0946. E-mail address: [email protected] (R.A. Ferriani).
follicular phase, however, there is a gradual increase in serum estradiol 17b concentration associated with a reduction in FSH levels. Despite this reduction in FSH, the growing follicle continues to mature, indicating that specific functional changes occur in the developing follicle that make it less dependent on FSH, while its dependence on LH increases. Since there is expression of LH receptors on the granulosa cells, almost all the physiological action of FSH on these cells, including stimulation of the aromatase system, could be performed by LH [4]. It has been suggested that LH may exert a deleterious effect on controlled ovarian stimulation, and some authors prefer recombinant drugs that essentially contain FSH [5]. In contrast, some recent studies have demonstrated an effective and positive role of LH in ovulation induction. Several recent reports have correlated low LH levels in the follicular phase with an inadequate effect on controlled ovarian
0301-2115/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2006.05.025
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stimulation [6,7]. LH supplementation for patients with hypogonadotropic hypogonadism is known to be of great importance for the optimization of the results of controlled ovarian stimulation [8,9]. In other studies, high recombinant LH (rLH) doses were tested in the late follicular phase to assess the reduction of excessive folliculogenesis in patients with hypogonadotropic hypogonadism [10] and in patients with polycystic ovary syndrome (PCOS) [11]. In both studies, this rLH dose was able to limit the development of minor antral follicles. LH alone may support estradiol 17b secretion at the end of the follicular phase in the absence of FSH replacement [12]. Recently, Filicori et al. [13] observed that, after priming with FSH, LH activity could be obtained with low doses of human chorionic gonadotropin (hCG), favoring to the stimulation of larger follicles and supporting them while FSH administration was reduced or discontinued. The occurrence of smaller follicles in the pre-ovulatory stage was significantly reduced, what could reduce the chance of developing ovarian hyperstimulation syndrome [14]. There was also a reduction in the FSH dose used, with a consequent reduction of the cost of treatment [13]. Thus, these regimens can have a profound impact on the optimization and safety of induced cycles. The application of this ovulation regimen to assisted reproduction techniques may combine efficacy and lower costs related to medication. The first pregnancy by ICSI obtained with low-dose hCG alone in the late controlled ovarian stimulation stages was reported recently [15], and this regimen reduced recombinant FSH consumption and was associated with a reduced number of small pre-ovulatory follicles [16]. Thus, the objective of the present study was to compare, in controlled ovarian stimulation cycles initially stimulated with rFSH, the exclusive use of hCG in the late follicular phase to the use of FSH alone (rFSH) or LH/FSH containing preparations (hMG) in terms of optimization of the cycle and better safety. Optimization was assessed on the basis of the gonadotropin doses used and of treatment cost. Safety was assessed on the basis of the reduction of follicles smaller than 10 mm and the endocrine outcome.
2. Materials and methods 2.1. Study population Fifty-one infertile women aged 25–35 years in good general health were selected at the Marital Infertility Outpatient Clinic of the University Hospital, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo (HCFMRP-USP). The patients had regular menstrual cycles, normal body mass index (BMI) of 20–25 kg/m2 and infertility due to tubal factor, infertility of no apparent cause and/or moderate or severe male factor (changes in
sperm numbers, motility or morphology leading to a final concentration of five million or less motile, progressive and normal sperm after sperm washing). ICSI was performed in all cases. Exclusion criteria were presence of PCOS, reduced ovarian function (FSH of more than 10 IU/mL) during the early follicular phase, endometriosis or uterine myomas, use of an injectable hormonal contraceptive up to 6 months before stimulation, a history of poor ovarian response to controlled ovarian stimulation, concomitant uterine alterations or absence of one ovary. 2.2. Protocol The study was approved by the Research Ethics Committee of HCFMRP-USP and by the National Council of Research Ethics (CONEP). All volunteers gave written informed consent to participate. The patients who filled the inclusion criteria were submitted to inhibition of the natural cycle with a low-dose oral contraceptive administered on the first the previous menstruation cycle and discontinued five days before the beginning of stimulation. aGnRH (leuprolide acetate: Lupron kit1, Abbott S/A Societe´ Francaise de Laboratories, Paris, France), 0.5 mg/day, was also used for inhibition starting ten days before the beginning of induction and continued until the day preceding the pre-ovulatory injection of hCG. Patients were randomly assigned through computer randomization to three treatment groups of 17 each. The controlled ovarian stimulation was divided into two phases. In the first phase, all the groups were supplemented with 200 IU rFSH (Puregon1, NV Organon, Holland) administered daily subcutaneously on the first days of induction until the dominant follicles reached 12– 13 mm in mean diameter. In the second phase, the patients were divided into hCG, hMG and rFSH groups. The hCG group received daily doses of 200 IU hCG (Profasi1 HP, Laboratoires Serono S.A., L’Ouriettaz, Switzerland) intramuscularly (IM) until the presence of follicles 18– 19 mm in diameter was detected. For hCG administration, 2000 IU were diluted in 10.0 mL 0.9% physiological saline and 1.0 mL of the solution was injected. The patients in the hMG group and rFSH group received daily IM injections of 225 IU hMG (Menogon1; Ferring Gmbh, Witland, Kiel, Germany) or daily SC 200 IU rFSH (Puregon1), respectively, until the parameters for preovulatory hCG were reached. When the desired follicle size was reached, 10,000 IU hCG were administered IM (Choragon1; Ferring Gmbh, Witland, Kiel, Germany) to all groups. The gonadotropins were administered to all women between 18:00 and 20:00 h. Oocyte retrieval was performed 36 h after pre-ovulatory hCG injection. Metaphase II oocytes were fertilized by ICSI according to previously described techniques [16] and embryo transfer was performed 72 h after retrieval.
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The luteal phase was supplemented with 90 mg progesterone gel (8% Crinone1, Fleet Laboratories Limited, Watford, UK, for Columbia Laboratories, Livingston, US, imported and distributed by Serono Produtos Farmaceˆuticos Ltda., Sa˜o Paulo, Brazil) administered daily by the vaginal route from the day of oocyte retrieval. The fertilization rate was defined as the ratio of the number of embryos formed to the number of oocytes inseminated. The implantation rate was defined as the ratio of the number of gestational sacs visualized by transvaginal pelvic ultrasound to the number of embryos transferred. The pregnancy rate was calculated by dividing the number of clinical pregnancies detected by the number of transfers performed (pregnancy rate/transfer) or by the number of punctures (pregnancy rate/puncture). 2.3. Monitoring The cycle was monitored by transvaginal pelvic ultrasound with an ATL–HDI 3000 instrument (ATL, Bothell, WA, USA), using a sectorial 5–9 MHz probe. The first ultrasound was performed on day 2 or 3 of the cycle, when the patient came to the outpatient clinic to start controlled ovarian stimulation, the second was performed on day 7 of induction, followed by examination on alternate days or daily until the day of pre-ovulatory hCG administration. Blood samples were collected on the days of basal transvaginal pelvic ultrasound (1st sample), of pre-ovulatory hCG injection (2nd sample) and of oocyte retrieval (3rd sample). Estradiol 17b (E2), progesterone (P) and testosterone (T) were determined in all samples and FSH, LH and PRL were also determined in the first one. LH and hCG were determined in the second sample. 2.4. Hormonal assays Hormone measurements were performed by chemiluminescence using an IMMULITE1 DPC-MED LAB instrument (Diagnostic Products Corporation–DPC, Los Angeles, CA, USA). The same kit was used for each specific hormone (IMMULITE1 Progesterone, IMMULITE1 Estradiol, IMMULITE1 FSH, IMMULITE1 LH, IMMULITE1 PRL, and IMMULITE1 hCG, all from DPC). T was determined by radioimmunoassay with the Tri Carb 2100 TR-Packard1 scintigraphy apparatus. The minimum detectable dose (MDD) was 0.1 mIU/mL for FSH, 0.05 mIU/mL for LH, 0.16 mIU/mL for hCG, 0.16 ng/mL for PRL, 15.0 pg/mL for E2, 0.2 ng/mL for P, and 20.0 ng/dL for T. 2.5. Statistical analysis The primary outcome measure was the amount of gonadotropins needed to achieve comparable levels of folliculogenesis and number of small (<10 mm diameter), medium (10–14 mm diameter) and large growing follicles (>14 mm diameter) measured during controlled ovarian
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stimulation. Secondary outcome measures included preovulatory serum concentrations of FSH, E2, P, and T, number of oocytes retrieved, fertilization, implantation rate, and pregnancy rate and total cost of treatment per embryo transferred. Sample size was calculated with the GraphPad Stat Mate software (Graphpad Software, San Diego, CA, USA). Using a two-tailed test with a 0.05 level of significance and a statistical power of 95%, we would have been able to detect a reduction of 50% or greater in the median of number of follicles smaller than 10 mm on the pre-ovulatory day with a sample of 16 patients per group. Data were analyzed statistically using the GraphPad Prism 3.01 software. The variables are reported as mean S.E. Comparisons among the three groups were carried out using analysis of variance (ANOVA), followed by the Tukey test when a significant difference was detected. The Kruskal–Wallis non-parametric test, followed by the Mann–Whitney test when there was a significant difference, was used for data that did not follow a normal distribution. Among-group differences in qualitative variables were assessed by with the x2 method.
3. Results The baseline patient characteristics of the three groups are shown in Table 1. There were no significant differences in parity, rate of abortion prior to treatment or time of infertility (data not shown) among the groups studied. No treatment cycle was cancelled after the patient entered the protocol. All patients were submitted to oocyte retrieval, but no embryos were obtained for two patients in the hMG group and for two patients in the rFSH group. The clinical and endocrine results of treatment are shown in Table 2. There was no difference among groups regarding the duration of treatment. The total rFSH dose administered was significantly higher for the rFSH group than for the hMG and hCG groups. Pre-ovulatory P and hCG levels were significantly higher in the hCG group than in the other two groups. Fourteen patients (82%) in the hCG group, 11 patients (65%) in the rFSH group, and 7 patients (41%) in the hMG group presented serum P levels higher than 1.5 ng/ mL on the day of pre-ovulatory hCG (hMG and rFSH progesterone levels were both lower than in the hCG group). Pre-ovulatory and oocyte retrieval’s day dosage of LH, E2, and T did not differ among the groups. Fig. 1 illustrates the profiles of E2, P, and T levels during the study. One patient each from the hCG and hMG groups presented moderate ovarian hyperstimulation syndrome, a phenomenon that had occurred in both of them in a previous induction cycle. The pattern of ultrasound-detected ovarian follicles during treatment in all patients is shown in Fig. 2. The number of large (diameter >14 mm), medium (diameter 10– 14 mm), and small follicles (diameter <10 mm) did not differ significantly among the three groups along the study.
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Table 1 Baseline characteristics in the three treatment groups hCG group (n = 17)
hMG group (n = 17)
rFSH group (n = 17)
Age (years) BMI (kg/m2) Menstrual cycle duration (median-days)
30.1 22.2 0.6 30 (25–35)
29.4 22.8 0.6 28 (25–35)
29.0 22.9 0.6 30 (28–35)
Infertility cause (%) Male factor Tubal factor Association
83 11 6
72 17 11
77 9 14
Mean ovarian volume (cm3) LH (mIU/mL) FSH (mIU/mL) PRL (ng/mL) T (ng/dL)
6.9 0.6 0.9 0.2 3.5 0.3 10.1 1.1 54.1 4.3
6.5 0.6 1.0 0.3 2.9 0.4 12.0 1.6 42.0 7.1
6.0 0.4 1.20 0.3 3.0 0.4 9.3 1.8 50.9 7.0
Data are expressed as mean S.E.; BMI, body mass index; LH, luteinizing hormone; FSH, follicle stimulation hormone; PRL, prolactin; T, testosterone. Table 2 Clinical and hormonal results of gonadotropin administration in the three treatment groups
Days Total Total Total
of gonadotropin administration—median rFSH dose received (IU) hCG dose received (IU) hMG dose received (IU)
hCG group (n = 17)
hMG group (n = 17)
rFSH group (n = 17)
10 (8–11) 1244 46a 612 58
10 (7–13) 1256 48 a
9 (7–13) 1929 73 b
614.71 76
Hormone levels on the pre-ovulatory hCG day hCG (mIU/mL) LH (mIU/mL) E2 (pg/mL) P (ng/mL) T (ng/dL)
17.8 3.1 b 1.3 0.3 3238 503 2.6 0.3 c 104.2 7.4
1.5 0.1 a 1.5 0.3 2164 308 1.5 0.1 d 79.6 12.8
All <1.0a 1.5 0.3 2057 252 2.0 0.3 d 97.5 10.4
No. of patients with P > 1.5 ng/mL
14c
7d
11
Hormone levels on OR day E2 (pg/mL) P (ng/mL) T (ng/dL)
1104 114 10.5 1.2 128.2 15.5
971 128 12.6 2.1 109.1 13.4
1514 223 17.7 3.6 116.0 11.0
Incidence of OHSS
1 of 17
1 of 17
None
Data are expressed as mean S.E.; E2, estradiol; P, progesterone; T, testosterone; OR, oocyte retrieval; ET, embryo transfer; OHSS, ovarian hyperstimulation syndrome; bp < 0.001 related to ‘‘a’’; cp < 0.05 related to ‘‘d’’.
The results of the fertilization process are shown in Table 3. Three miscarriages occurred in the hCG group, as opposed to one in the rFSH group and to none in the hMG group. Fifty-seven percent of the pregnancies were singleton gestations (three in the hCG group, two in the hMG group, and three in the rFSH group). The incidence of twin pregnancies was 29% (three in the hCG group and one in the hMG group) and the incidence of triplet pregnancies was 14% (two in the hMG group) The total cost of treatment was significantly lower in the hCG group than in the hMG and rFSH groups, but the cost per embryo transferred did not differ among groups.
4. Discussion The present results show that controlled ovarian stimulation cycles initially stimulated with rFSH followed
by exclusive hCG in the late follicular phase exhibited the same ovarian response pattern compared to cycles stimulated with hMG and rFSH in the same phase. The duration of treatment and the follicular growth pattern were similar in the three treatment groups. Controversy still exists about the gonadotropin preparations that provide the best results in controlled ovarian stimulation, especially in view of the commercial interests involved. Urinary gonadotropins started to be used in the early 1960s for ovulation induction and continued to be used until the introduction of rFSH on the pharmaceutical market, and have been used for ovarian stimulation since the 1980 decade. In addition to the difficulty in their collection from urine, urinary gonadotropins have several other disadvantages such as contamination with more than 95% ‘‘nonFSH’’ proteins, a high incidence of local allergic reactions, the heterogeneity of the different lots, and the presence of LH. The LH activity found in hMG derives from the
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Fig. 1. Mean profile of serum levels of estradiol 17b (E2), progesterone (P) and testosterone (T) in the three groups throughout the study: day zero (D 0), day of pre-ovulatory hCG (D hCG), and day of oocyte retrieval (OR). * p < 0.05 (serum P in the hCG group was significantly higher than in the hMG and rFSH groups).
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Fig. 2. Number (median S.E.) of small (<10 mm diameter, panel (A), medium (10–14 mm diameter, panel (B), and large growing follicles (>14 mm diameter, panel (C), measured during controlled ovarian stimulation (basal day, D 0; day that follicles with 13 mm diameter were detected, end of first phase; day of hCG administration for ovulation induction, day of hCG).
Table 3 Results of the fertilization process in the three treatment groups of patients submitted to controlled ovarian stimulation and ICSI
No. of punctured follicles No. of retrieved oocytes No. of embryos formed No. of embryos transferred No. of transfers Fertilization rate (%) Implantation rate (%) Pregnancy rate/transfer (%) Pregnancy rate/puncture (%) Abortion Total cost per embryo transferred (US$) Total cost (US$)
hCG group (n = 17)
hMG group (n = 17)
rFSH group (n = 17)
20.7 3.2 11.6 1.5 6.3 0.9 2.6 0.2 17.0 71.6 70.4 53.0 53.0 3/9 280 32 650 24a
19.3 3.0 10.9 1.4 4.8 0.9 2.4 0.3 15.0 59.1 80.0 33.0 29.0 0/5 333 50 788 33b
21.6 2.0 10.2 0.9 4.4 0.6 2.5 0.2 15.0 55.4 38.7 35.0 31.8 1/4 327 23 885 69 c
Data are expressed as mean S.E.; ap < 0.05 related to ‘‘b’’ and ‘‘c’’.
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combination of LH and hCG and the exact amounts of each of these hormones present in gonadotropin are not declared by the manufacturing laboratories. If insufficient LH quantities are present during industrial production, hCG is added in order to produce a drug conforming with the standards required by the pharmaceutical industry [17]. Several studies have analyzed the benefits of continuous use of exclusive FSH versus FSH in combination with LH activity in controlled ovarian stimulation [18–21]. It should be pointed out, however, that no consensus has been reached regarding the ideal amount of exogenous LH needed for controlled ovarian stimulation schemes. In contrast to the cited reports, which assessed the role of LH throughout follicular development, current concepts regarding the physiological role of FSH and LH in folliculogenesis consider LH to play an essential role in the final stages of follicular maturation [4]. In this context, the present study was conducted in order to assess the activity of exclusive LH compared to hMG and to rFSH in the late follicular phase, i.e., when the mean follicular diameter reaches 13 mm after ovarian stimulation with rFSH. This LH activity was administered in the form of lowdose hCG. hCG selectively binds to LH receptors and exerts the same actions as LH, but with an approximately six-fold higher potency. When considering hCG and rLH as LH activity, we chose to use hCG because its longer decay period ensures a more stable stimulation of LH receptors over the 24 h intervals between hormone administrations. Previous studies have demonstrated that the use of a low hCG dose (200 IU/day) was sufficient to stimulate folliculogenesis after priming with rFSH and to induce pregnancy [13,15,16,22,23]. Our study, however, compared for the first time the use of low-dose hCG with the use of hMG or rFSH in the late follicular phase and permitted us to compare the LH activity associated with FSH (hMG group) and FSH alone (rFSH group) with the activity of selective LH (hCG group), and no significant differences were found. In the present study, pituitary suppression was performed with daily administered aGnRH, with low, but not fully suppressed basal LH levels in both groups. It should be pointed out that aGnRH usually does not result in full LH elimination and it is accepted that less than 1.0% of follicular LH receptors need to be occupied in order to obtain a maximum steroidogenic response. Maximal thecal cell stimulation can be obtained at LH concentrations of 1.0 to 10.0 IU/L [5], as reported in a recent study on young oocyte donors submitted to blockade with aGnRH. The study demonstrated that the inclusion of exogenous LH activity, i.e. activity in the form of hMG, in the induction protocols had beneficial effects on the environment and on the oocyte quality of donors who presented LH concentrations <1.0 IU/L after pituitary suppression [24]. However, in the cited study, pituitary suppression was performed with depot aGnRH preceded by oral norethisterone, two hormones that are responsible for profound pituitary blockade.
In the present study, all patients in the three groups reached the final stage of follicular maturation. This was probably due to the fact that, in the control group, 13 mm follicles presented LH receptors responsive to hCG activity. Protocols that recommended the introduction of hCG after a fixed period of rFSH administration did not achieve the same success since some patients did not reach the final stages of follicular maturation [13]. Thus, our proposal of using the follicular size parameter to change the stimulation scheme and not a fixed number of days was effective, supporting the hypothesis of a more physiological role of LH in the final phase of stimulation. The growth pattern of follicles measuring <10, 10–14, and >14 mm in mean diameter along controlled ovarian stimulation and the pre-ovulatory serum concentrations of E2 and T did not differ among the three treatment groups. Similarly, there was no difference in the dose of gonadotropin needed to reach the final parameters of follicular maturation. Only one patient in each group presented ovarian hyperstimulation syndrome. Our initial hypothesis was that the number of follicles measuring less than 10 mm would be smaller in the hCG scheme, as suggested by Filicori et al. [13]. In the cited study, the effect of a low hCG dose was assessed after one week of controlled ovarian stimulation with rFSH (150 IU/day) in GnRH agonist-suppressed normoovulatory women. Four different protocols were tested: 150 IU/day of rFSH alone, 50 IU/day rFSH plus 50 IU/day hCG, 25 IU/day rFSH plus 100 IU/day hCG, and 200 IU/day hCG alone. No significant difference was detected among the four groups regarding the number of pre-ovulatory follicles measuring 10–14 and >14 mm or the pre-ovulatory concentration of estradiol. However, there was a reduction of pre-ovulatory ovarian follicles <10 mm in the patients who received hCG and the amount of rFSH necessary to reach the final parameters of maturation was progressively reduced with the increasing proportion of hCG administered. The decrease of smaller follicles was correlated with a lower chance of ovarian hyperstimulation syndrome [14]. These results indicate a combination of safety and lower costs because of the reduced dose of gonadotropin used. However, the results of the present study regarding the number of follicles <10 mm and the gonadotropin dose needed for follicular maturation differ from those reported by Filicori et al. [13]. On the other hand, our study agrees with that of Filicori et al. [13,15,16] by demonstrating that stimulation with hCG in the final follicular phase is effective and does not require exogenous FSH, with a consequent reduction of total treatment costs. Regarding the results of the assisted reproduction process, such as number of oocytes retrieved, embryos formed and transferred, and fertilization, implantation and pregnancy rates, the present series is insufficient for an analysis with a significant test power. However, it is tempting to speculate if the difference in pregnancy rate would be significant with a larger series. Similarly, in view
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of the above results, we may attribute a positive role to LH administered in the late follicular phase regarding oocyte quality. Recent studies [16,25] obtained similar results when assessing the use of selective hCG in the late follicular phase in a larger series. There are also reports of successful controlled ovarian stimulation cycles in normoovulatory women with the use of rLH alone in the late follicular phase after the initial stimulation with rFSH [26]. An interesting feature of the present study was that, despite the significantly high P levels on the pre-ovulatory hCG day in the hCG group compared to the hMG and rFSH groups, there was no deleterious effect on the final results. It has been reported that 2–35% of controlled ovarian stimulation cycles suppressed with aGnRH present serum P elevation during treatment [27]. The clinical impact of early luteinization, demonstrated by a sudden increase in P (with a cut-off level ranging from 0.5 to 2.0 ng/mL in the literature) on the day preovulatory hCG day continues to be controversial. While some studies have reported that a serum increase of this hormone during the pre-ovulatory period has a negative impact on pregnancy rates after assisted reproduction [28], others disagree and consider the difference in serum P levels on the day of pre-ovulatory hCG administration not to be correlated with clinical pregnancy rate in assisted reproduction [27,29]. In an oocyte donation program, early luteinization was associated with an increase in pregnancy rates [30]. Recently, Martinez et al. [31] demonstrated by means of the receiver–operating characteristic (ROC) curve that serum P concentration on the hCG day cannot predict the occurrence of pregnancy in assisted reproduction cycles using aGnRH and gonadotropins. These findings also permit us to propose that the pre-ovulatory elevation in P does not depend on LH. No significant differences regarding pregnancy or abortion rates were observed between groups with P levels lower or higher than 0.9 ng/mL.
5. Conclusions The present study demonstrated successful results of assisted reproduction processes with controlled ovarian stimulation protocols by using selective LH activity in the late follicular phase. With an ovarian response pattern similar to that obtained with hMG and with rFSH, it is possible to use a protocol of significantly lower cost. The protocol evaluated (hCG group) produced pregnancy rates similar to those produced in the hMG and rFSH groups even though serum P levels were significantly higher on the hCG day. Further studies conducted on larger series to assess the use of hCG in the late follicular phase are needed in order to demonstrate that efficacy and safety are comparable to those obtained in cycles supplemented with hMG in the same phase or with rFSH alone. Analysis of the oocyte quality of patients submitted to induction with hCG at the end of the follicular phase may contribute to the investigation of the role of LH during this period.
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6. Condensation Ovulation induction with hCG during the late follicular phase induces the same response pattern as that obtained with hMG or rFSH during the same phase.
Acknowledgments We wish to thank the Center of Quantitative Methods (CEMEQ) of FMRP-USP for collaboration with the statistical analysis.
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[14] Navot D, Relou A, Birkenfeld A, Rabinowitz R, Brzezinski A, Margalioth EJ. Risk factors and prognostic variables in the ovarian hyperstimulation syndrome. Am J Obstet Gynecol 1988;159:210–5. [15] Filicori M, Cognigni GE, Taraborrelli S, Parmegiani L, Bernardi S, Ciampaglia W. Intracytoplasmic sperm injection pregnancy after low dose human chorionic gonadotropin alone to support ovarian folliculogenesis. Fertil Steril 2002;78:414–6. [16] Filicori M, Cognigni GE, Gamberini E, Parmegiani L, Troilo E, Roset B. Efficacy of low-dose human chorionic gonadotropin alone to complete controlled ovarian stimulation. Fertil Steril 2005;84:394– 401. [17] Stockman PG, de Leeuw R, van den Wijngaard HA, Kloosterboer HJ, Vemer HM, Sander AL. Human chorionic gonadotropin in commercial human menopausal gonadotropin preparations. Fertil Steril 1993;60:175–80. [18] Agrawal R, Holmes J, Jacobs HS. Follicle-stimulating hormone or human menopausal gonadotropin for ovarian stimulation in in vitro fertilization cycles: a meta-analysis. Fertil Steril 2000;73:338–43. [19] Ng EHY, Lau EYL, Yeung WSB, Ho PC. HMG is as good as recombinant human FSH in terms of oocyte and embryo quality: a prospective randomized trial. Hum Reprod 2001;16:319–25. [20] The European and Israeli Study Group on Highly Purified Menotropin versus Recombinant Follicle-Stimulating Hormone. Efficacy and safety of highly purified menotropin versus recombinant folliclestimulating hormone in in vitro fertilization/intracytoplasmic sperm injection cycles: a randomized, comparative trial. Fertil Steril 2002;78:520–8. [21] Kilani Z, Dakkak A, Ghunaim S, et al. A prospective, randomized, controlled trial comparing highly purified hMG with recombinant FSH in women undergoing ICSI: ovarian response and clinical outcomes. Hum Reprod 2003;18:1194–9. [22] Lee KL, Couchman GM, Walmer1 DK. Successful pregnancies in patients with estrogenic anovulation after low-dose human chorionic gonadotropin therapy alone following hMG for controlled ovarian hyperstimulation. J Assist Reprod Gen 2005;22:37–40.
[23] Branigan EF, Estes A. Use of micro-dose human chorionic gonadotropin (hCG) after clomiphene citrate (CC) to complete folliculogenesis in previous CC-resistant anovulation. Am J Obstet Gynecol 2005;192:1890–6. [24] Tesarik J, Mendonza C. Effects of exogenous LH administration during ovarian stimulation of pituitary down-regulated young oocyte donors on oocyte yield and developmental competence. Hum Reprod 2002;17:3129–37. [25] Filicori M, Cognigni GE, Parmegiani L, et al. Efficacy of low-dose hCG in ovarian stimulation. In: Abstracts of the 20th annual meeting of the ESHRE. 2004. p. i119–20. [26] Hazout A, Junca AM, Bacrie PC. FSH coasting combined with recombinant LH: a novel stimulation approach for ART. Fertil Steril 2003;80:S8. [27] Ubaldi F, Camus M, Smitz J, Bennink HC, Van Steirteghem A, Devroey P. Premature luteinization in in vitro fertilization cycles using gonadotropin-releasing hormone agonist (GnRH-a) and recombinant follicle-stimulating hormone (FSH) and GnRH-a and urinary FSH. Fertil Steril 1996;66:275–80. [28] Shulman A, Ghetler Y, Beyth Y, Ben-Nun I. The significance of an early (premature) rise of plasma progesterone in in vitro fertilization cycles induced by a ‘‘long protocol’’ of gonadotropin releasing hormone analogue and human menopausal gonadotropins. J Assist Reprod Gen 1996;13:207–11. [29] Hofmann GE, Khoury J, Jonhson CA, Thie J, Scott Jr RT. Premature luteinization during controlled ovarian hyperstimulation for in vitro fertilization-embryo transfer has no impact on pregnancy outcome. Fertil Steril 1996;66:980–8. [30] Legro RS, Ary BA, Paulson RJ, Stanczyk FZ, Sauer MV. Premature luteinization as detected by elevated serum progesterone is associated with a higher pregnancy rate in donor oocyte in vitro fertilization. Hum Reprod 1993;8:1506–11. [31] Martinez F, Coroleu B, Clua E, et al. Serum progesterone concentrations on the day of HCG administration cannot predict pregnancy in assisted reproduction cycles. Reprod Biomed Online 2003;8:183–90.
Controlled ovarian stimulation with exclusive FSH followed by stimulation with hCG alone, FSH alone or hMG Mariana K.O. Gomes, Carolina S. Vieira, Marcos D. Moura, Luiz A. Manetta, Stael P. Leite, Rosana M. Reis, Rui A. Ferriani * Department of Gynaecology and Obstetrics of the University of Sa˜o Paulo Ribeira˜o Preto School of Medicine, Ribeira˜o Preto, Brazil Received 27 September 2005; received in revised form 16 April 2006; accepted 17 May 2006
Abstract Objective: To assess if low-dose hCG is similar to hMG and to rFSH in the late follicular phase. Study design: In a prospective randomized controlled trial, 51 patients undergoing controlled ovarian stimulation received ovarian priming with rFSH and then received hCG (200 IU/day) (hCG group, n = 17), hMG (225 IU/day) (hMG group, n = 17) or rFSH (200 IU/day) (FSH group, n = 17) in the late stage of follicular development. Parameters of follicular response and serum estradiol, progesterone and testosterone levels were assessed. Results: Pre-ovulatory ovarian follicle occurrence and length of treatment were similar among the three treatment groups. Serum progesterone level on the day of pre-ovulatory hCG was significantly higher in the hCG group than in the hMG or rFSH group. Clinical pregnancy rates were similar for all groups. The total cost of treatment was significantly lower for the hCG group than for the groups supplemented with hMG or rFSH. Conclusions: LH in the form of low-dose hCG during the late follicular phase induced the same follicular pattern as hMG and rFSH after ovulation induction. The procedure using hCG produced pregnancy rates similar to those obtained using hMG and rFSH, even though the patients showed higher serum progesterone levels on the hCG day. # 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Ovulation induction; Human chorionic gonadotropin; Human menopausal gonadotropin; Recombinant FSH; Luteinizing hormone
1. Introduction The importance of luteinizing hormone (LH) in the follicular phase and its role in ovulation induction are the subject of extensive debate. The optimal LH levels in assisted reproduction protocols and the drugs to be used for this purpose are still controversial topics [1–3]. Early follicular phase prevalence of follicle stimulating hormone (FSH) is responsible for follicle recruitment and the initial stimulation of the growth and development of granulosa cells in the pre-ovulatory follicle. In the middle * Corresponding author at: Av. Bandeirantes 3900, Campus Universita´rio, Departamento de Ginecologia e Obstetrı´cia da Faculdade de Medicina de Ribeira˜o Preto/USP, CEP 14049-900 Ribeira˜o Preto, SP Brasil. Tel.: +55 16 3602 2818; fax: +55 16 3633 0946. E-mail address: [email protected] (R.A. Ferriani).
follicular phase, however, there is a gradual increase in serum estradiol 17b concentration associated with a reduction in FSH levels. Despite this reduction in FSH, the growing follicle continues to mature, indicating that specific functional changes occur in the developing follicle that make it less dependent on FSH, while its dependence on LH increases. Since there is expression of LH receptors on the granulosa cells, almost all the physiological action of FSH on these cells, including stimulation of the aromatase system, could be performed by LH [4]. It has been suggested that LH may exert a deleterious effect on controlled ovarian stimulation, and some authors prefer recombinant drugs that essentially contain FSH [5]. In contrast, some recent studies have demonstrated an effective and positive role of LH in ovulation induction. Several recent reports have correlated low LH levels in the follicular phase with an inadequate effect on controlled ovarian
0301-2115/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2006.05.025
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stimulation [6,7]. LH supplementation for patients with hypogonadotropic hypogonadism is known to be of great importance for the optimization of the results of controlled ovarian stimulation [8,9]. In other studies, high recombinant LH (rLH) doses were tested in the late follicular phase to assess the reduction of excessive folliculogenesis in patients with hypogonadotropic hypogonadism [10] and in patients with polycystic ovary syndrome (PCOS) [11]. In both studies, this rLH dose was able to limit the development of minor antral follicles. LH alone may support estradiol 17b secretion at the end of the follicular phase in the absence of FSH replacement [12]. Recently, Filicori et al. [13] observed that, after priming with FSH, LH activity could be obtained with low doses of human chorionic gonadotropin (hCG), favoring to the stimulation of larger follicles and supporting them while FSH administration was reduced or discontinued. The occurrence of smaller follicles in the pre-ovulatory stage was significantly reduced, what could reduce the chance of developing ovarian hyperstimulation syndrome [14]. There was also a reduction in the FSH dose used, with a consequent reduction of the cost of treatment [13]. Thus, these regimens can have a profound impact on the optimization and safety of induced cycles. The application of this ovulation regimen to assisted reproduction techniques may combine efficacy and lower costs related to medication. The first pregnancy by ICSI obtained with low-dose hCG alone in the late controlled ovarian stimulation stages was reported recently [15], and this regimen reduced recombinant FSH consumption and was associated with a reduced number of small pre-ovulatory follicles [16]. Thus, the objective of the present study was to compare, in controlled ovarian stimulation cycles initially stimulated with rFSH, the exclusive use of hCG in the late follicular phase to the use of FSH alone (rFSH) or LH/FSH containing preparations (hMG) in terms of optimization of the cycle and better safety. Optimization was assessed on the basis of the gonadotropin doses used and of treatment cost. Safety was assessed on the basis of the reduction of follicles smaller than 10 mm and the endocrine outcome.
2. Materials and methods 2.1. Study population Fifty-one infertile women aged 25–35 years in good general health were selected at the Marital Infertility Outpatient Clinic of the University Hospital, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo (HCFMRP-USP). The patients had regular menstrual cycles, normal body mass index (BMI) of 20–25 kg/m2 and infertility due to tubal factor, infertility of no apparent cause and/or moderate or severe male factor (changes in
sperm numbers, motility or morphology leading to a final concentration of five million or less motile, progressive and normal sperm after sperm washing). ICSI was performed in all cases. Exclusion criteria were presence of PCOS, reduced ovarian function (FSH of more than 10 IU/mL) during the early follicular phase, endometriosis or uterine myomas, use of an injectable hormonal contraceptive up to 6 months before stimulation, a history of poor ovarian response to controlled ovarian stimulation, concomitant uterine alterations or absence of one ovary. 2.2. Protocol The study was approved by the Research Ethics Committee of HCFMRP-USP and by the National Council of Research Ethics (CONEP). All volunteers gave written informed consent to participate. The patients who filled the inclusion criteria were submitted to inhibition of the natural cycle with a low-dose oral contraceptive administered on the first the previous menstruation cycle and discontinued five days before the beginning of stimulation. aGnRH (leuprolide acetate: Lupron kit1, Abbott S/A Societe´ Francaise de Laboratories, Paris, France), 0.5 mg/day, was also used for inhibition starting ten days before the beginning of induction and continued until the day preceding the pre-ovulatory injection of hCG. Patients were randomly assigned through computer randomization to three treatment groups of 17 each. The controlled ovarian stimulation was divided into two phases. In the first phase, all the groups were supplemented with 200 IU rFSH (Puregon1, NV Organon, Holland) administered daily subcutaneously on the first days of induction until the dominant follicles reached 12– 13 mm in mean diameter. In the second phase, the patients were divided into hCG, hMG and rFSH groups. The hCG group received daily doses of 200 IU hCG (Profasi1 HP, Laboratoires Serono S.A., L’Ouriettaz, Switzerland) intramuscularly (IM) until the presence of follicles 18– 19 mm in diameter was detected. For hCG administration, 2000 IU were diluted in 10.0 mL 0.9% physiological saline and 1.0 mL of the solution was injected. The patients in the hMG group and rFSH group received daily IM injections of 225 IU hMG (Menogon1; Ferring Gmbh, Witland, Kiel, Germany) or daily SC 200 IU rFSH (Puregon1), respectively, until the parameters for preovulatory hCG were reached. When the desired follicle size was reached, 10,000 IU hCG were administered IM (Choragon1; Ferring Gmbh, Witland, Kiel, Germany) to all groups. The gonadotropins were administered to all women between 18:00 and 20:00 h. Oocyte retrieval was performed 36 h after pre-ovulatory hCG injection. Metaphase II oocytes were fertilized by ICSI according to previously described techniques [16] and embryo transfer was performed 72 h after retrieval.
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The luteal phase was supplemented with 90 mg progesterone gel (8% Crinone1, Fleet Laboratories Limited, Watford, UK, for Columbia Laboratories, Livingston, US, imported and distributed by Serono Produtos Farmaceˆuticos Ltda., Sa˜o Paulo, Brazil) administered daily by the vaginal route from the day of oocyte retrieval. The fertilization rate was defined as the ratio of the number of embryos formed to the number of oocytes inseminated. The implantation rate was defined as the ratio of the number of gestational sacs visualized by transvaginal pelvic ultrasound to the number of embryos transferred. The pregnancy rate was calculated by dividing the number of clinical pregnancies detected by the number of transfers performed (pregnancy rate/transfer) or by the number of punctures (pregnancy rate/puncture). 2.3. Monitoring The cycle was monitored by transvaginal pelvic ultrasound with an ATL–HDI 3000 instrument (ATL, Bothell, WA, USA), using a sectorial 5–9 MHz probe. The first ultrasound was performed on day 2 or 3 of the cycle, when the patient came to the outpatient clinic to start controlled ovarian stimulation, the second was performed on day 7 of induction, followed by examination on alternate days or daily until the day of pre-ovulatory hCG administration. Blood samples were collected on the days of basal transvaginal pelvic ultrasound (1st sample), of pre-ovulatory hCG injection (2nd sample) and of oocyte retrieval (3rd sample). Estradiol 17b (E2), progesterone (P) and testosterone (T) were determined in all samples and FSH, LH and PRL were also determined in the first one. LH and hCG were determined in the second sample. 2.4. Hormonal assays Hormone measurements were performed by chemiluminescence using an IMMULITE1 DPC-MED LAB instrument (Diagnostic Products Corporation–DPC, Los Angeles, CA, USA). The same kit was used for each specific hormone (IMMULITE1 Progesterone, IMMULITE1 Estradiol, IMMULITE1 FSH, IMMULITE1 LH, IMMULITE1 PRL, and IMMULITE1 hCG, all from DPC). T was determined by radioimmunoassay with the Tri Carb 2100 TR-Packard1 scintigraphy apparatus. The minimum detectable dose (MDD) was 0.1 mIU/mL for FSH, 0.05 mIU/mL for LH, 0.16 mIU/mL for hCG, 0.16 ng/mL for PRL, 15.0 pg/mL for E2, 0.2 ng/mL for P, and 20.0 ng/dL for T. 2.5. Statistical analysis The primary outcome measure was the amount of gonadotropins needed to achieve comparable levels of folliculogenesis and number of small (<10 mm diameter), medium (10–14 mm diameter) and large growing follicles (>14 mm diameter) measured during controlled ovarian
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stimulation. Secondary outcome measures included preovulatory serum concentrations of FSH, E2, P, and T, number of oocytes retrieved, fertilization, implantation rate, and pregnancy rate and total cost of treatment per embryo transferred. Sample size was calculated with the GraphPad Stat Mate software (Graphpad Software, San Diego, CA, USA). Using a two-tailed test with a 0.05 level of significance and a statistical power of 95%, we would have been able to detect a reduction of 50% or greater in the median of number of follicles smaller than 10 mm on the pre-ovulatory day with a sample of 16 patients per group. Data were analyzed statistically using the GraphPad Prism 3.01 software. The variables are reported as mean S.E. Comparisons among the three groups were carried out using analysis of variance (ANOVA), followed by the Tukey test when a significant difference was detected. The Kruskal–Wallis non-parametric test, followed by the Mann–Whitney test when there was a significant difference, was used for data that did not follow a normal distribution. Among-group differences in qualitative variables were assessed by with the x2 method.
3. Results The baseline patient characteristics of the three groups are shown in Table 1. There were no significant differences in parity, rate of abortion prior to treatment or time of infertility (data not shown) among the groups studied. No treatment cycle was cancelled after the patient entered the protocol. All patients were submitted to oocyte retrieval, but no embryos were obtained for two patients in the hMG group and for two patients in the rFSH group. The clinical and endocrine results of treatment are shown in Table 2. There was no difference among groups regarding the duration of treatment. The total rFSH dose administered was significantly higher for the rFSH group than for the hMG and hCG groups. Pre-ovulatory P and hCG levels were significantly higher in the hCG group than in the other two groups. Fourteen patients (82%) in the hCG group, 11 patients (65%) in the rFSH group, and 7 patients (41%) in the hMG group presented serum P levels higher than 1.5 ng/ mL on the day of pre-ovulatory hCG (hMG and rFSH progesterone levels were both lower than in the hCG group). Pre-ovulatory and oocyte retrieval’s day dosage of LH, E2, and T did not differ among the groups. Fig. 1 illustrates the profiles of E2, P, and T levels during the study. One patient each from the hCG and hMG groups presented moderate ovarian hyperstimulation syndrome, a phenomenon that had occurred in both of them in a previous induction cycle. The pattern of ultrasound-detected ovarian follicles during treatment in all patients is shown in Fig. 2. The number of large (diameter >14 mm), medium (diameter 10– 14 mm), and small follicles (diameter <10 mm) did not differ significantly among the three groups along the study.
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Table 1 Baseline characteristics in the three treatment groups hCG group (n = 17)
hMG group (n = 17)
rFSH group (n = 17)
Age (years) BMI (kg/m2) Menstrual cycle duration (median-days)
30.1 22.2 0.6 30 (25–35)
29.4 22.8 0.6 28 (25–35)
29.0 22.9 0.6 30 (28–35)
Infertility cause (%) Male factor Tubal factor Association
83 11 6
72 17 11
77 9 14
Mean ovarian volume (cm3) LH (mIU/mL) FSH (mIU/mL) PRL (ng/mL) T (ng/dL)
6.9 0.6 0.9 0.2 3.5 0.3 10.1 1.1 54.1 4.3
6.5 0.6 1.0 0.3 2.9 0.4 12.0 1.6 42.0 7.1
6.0 0.4 1.20 0.3 3.0 0.4 9.3 1.8 50.9 7.0
Data are expressed as mean S.E.; BMI, body mass index; LH, luteinizing hormone; FSH, follicle stimulation hormone; PRL, prolactin; T, testosterone. Table 2 Clinical and hormonal results of gonadotropin administration in the three treatment groups
Days Total Total Total
of gonadotropin administration—median rFSH dose received (IU) hCG dose received (IU) hMG dose received (IU)
hCG group (n = 17)
hMG group (n = 17)
rFSH group (n = 17)
10 (8–11) 1244 46a 612 58
10 (7–13) 1256 48 a
9 (7–13) 1929 73 b
614.71 76
Hormone levels on the pre-ovulatory hCG day hCG (mIU/mL) LH (mIU/mL) E2 (pg/mL) P (ng/mL) T (ng/dL)
17.8 3.1 b 1.3 0.3 3238 503 2.6 0.3 c 104.2 7.4
1.5 0.1 a 1.5 0.3 2164 308 1.5 0.1 d 79.6 12.8
All <1.0a 1.5 0.3 2057 252 2.0 0.3 d 97.5 10.4
No. of patients with P > 1.5 ng/mL
14c
7d
11
Hormone levels on OR day E2 (pg/mL) P (ng/mL) T (ng/dL)
1104 114 10.5 1.2 128.2 15.5
971 128 12.6 2.1 109.1 13.4
1514 223 17.7 3.6 116.0 11.0
Incidence of OHSS
1 of 17
1 of 17
None
Data are expressed as mean S.E.; E2, estradiol; P, progesterone; T, testosterone; OR, oocyte retrieval; ET, embryo transfer; OHSS, ovarian hyperstimulation syndrome; bp < 0.001 related to ‘‘a’’; cp < 0.05 related to ‘‘d’’.
The results of the fertilization process are shown in Table 3. Three miscarriages occurred in the hCG group, as opposed to one in the rFSH group and to none in the hMG group. Fifty-seven percent of the pregnancies were singleton gestations (three in the hCG group, two in the hMG group, and three in the rFSH group). The incidence of twin pregnancies was 29% (three in the hCG group and one in the hMG group) and the incidence of triplet pregnancies was 14% (two in the hMG group) The total cost of treatment was significantly lower in the hCG group than in the hMG and rFSH groups, but the cost per embryo transferred did not differ among groups.
4. Discussion The present results show that controlled ovarian stimulation cycles initially stimulated with rFSH followed
by exclusive hCG in the late follicular phase exhibited the same ovarian response pattern compared to cycles stimulated with hMG and rFSH in the same phase. The duration of treatment and the follicular growth pattern were similar in the three treatment groups. Controversy still exists about the gonadotropin preparations that provide the best results in controlled ovarian stimulation, especially in view of the commercial interests involved. Urinary gonadotropins started to be used in the early 1960s for ovulation induction and continued to be used until the introduction of rFSH on the pharmaceutical market, and have been used for ovarian stimulation since the 1980 decade. In addition to the difficulty in their collection from urine, urinary gonadotropins have several other disadvantages such as contamination with more than 95% ‘‘nonFSH’’ proteins, a high incidence of local allergic reactions, the heterogeneity of the different lots, and the presence of LH. The LH activity found in hMG derives from the
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Fig. 1. Mean profile of serum levels of estradiol 17b (E2), progesterone (P) and testosterone (T) in the three groups throughout the study: day zero (D 0), day of pre-ovulatory hCG (D hCG), and day of oocyte retrieval (OR). * p < 0.05 (serum P in the hCG group was significantly higher than in the hMG and rFSH groups).
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Fig. 2. Number (median S.E.) of small (<10 mm diameter, panel (A), medium (10–14 mm diameter, panel (B), and large growing follicles (>14 mm diameter, panel (C), measured during controlled ovarian stimulation (basal day, D 0; day that follicles with 13 mm diameter were detected, end of first phase; day of hCG administration for ovulation induction, day of hCG).
Table 3 Results of the fertilization process in the three treatment groups of patients submitted to controlled ovarian stimulation and ICSI
No. of punctured follicles No. of retrieved oocytes No. of embryos formed No. of embryos transferred No. of transfers Fertilization rate (%) Implantation rate (%) Pregnancy rate/transfer (%) Pregnancy rate/puncture (%) Abortion Total cost per embryo transferred (US$) Total cost (US$)
hCG group (n = 17)
hMG group (n = 17)
rFSH group (n = 17)
20.7 3.2 11.6 1.5 6.3 0.9 2.6 0.2 17.0 71.6 70.4 53.0 53.0 3/9 280 32 650 24a
19.3 3.0 10.9 1.4 4.8 0.9 2.4 0.3 15.0 59.1 80.0 33.0 29.0 0/5 333 50 788 33b
21.6 2.0 10.2 0.9 4.4 0.6 2.5 0.2 15.0 55.4 38.7 35.0 31.8 1/4 327 23 885 69 c
Data are expressed as mean S.E.; ap < 0.05 related to ‘‘b’’ and ‘‘c’’.
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combination of LH and hCG and the exact amounts of each of these hormones present in gonadotropin are not declared by the manufacturing laboratories. If insufficient LH quantities are present during industrial production, hCG is added in order to produce a drug conforming with the standards required by the pharmaceutical industry [17]. Several studies have analyzed the benefits of continuous use of exclusive FSH versus FSH in combination with LH activity in controlled ovarian stimulation [18–21]. It should be pointed out, however, that no consensus has been reached regarding the ideal amount of exogenous LH needed for controlled ovarian stimulation schemes. In contrast to the cited reports, which assessed the role of LH throughout follicular development, current concepts regarding the physiological role of FSH and LH in folliculogenesis consider LH to play an essential role in the final stages of follicular maturation [4]. In this context, the present study was conducted in order to assess the activity of exclusive LH compared to hMG and to rFSH in the late follicular phase, i.e., when the mean follicular diameter reaches 13 mm after ovarian stimulation with rFSH. This LH activity was administered in the form of lowdose hCG. hCG selectively binds to LH receptors and exerts the same actions as LH, but with an approximately six-fold higher potency. When considering hCG and rLH as LH activity, we chose to use hCG because its longer decay period ensures a more stable stimulation of LH receptors over the 24 h intervals between hormone administrations. Previous studies have demonstrated that the use of a low hCG dose (200 IU/day) was sufficient to stimulate folliculogenesis after priming with rFSH and to induce pregnancy [13,15,16,22,23]. Our study, however, compared for the first time the use of low-dose hCG with the use of hMG or rFSH in the late follicular phase and permitted us to compare the LH activity associated with FSH (hMG group) and FSH alone (rFSH group) with the activity of selective LH (hCG group), and no significant differences were found. In the present study, pituitary suppression was performed with daily administered aGnRH, with low, but not fully suppressed basal LH levels in both groups. It should be pointed out that aGnRH usually does not result in full LH elimination and it is accepted that less than 1.0% of follicular LH receptors need to be occupied in order to obtain a maximum steroidogenic response. Maximal thecal cell stimulation can be obtained at LH concentrations of 1.0 to 10.0 IU/L [5], as reported in a recent study on young oocyte donors submitted to blockade with aGnRH. The study demonstrated that the inclusion of exogenous LH activity, i.e. activity in the form of hMG, in the induction protocols had beneficial effects on the environment and on the oocyte quality of donors who presented LH concentrations <1.0 IU/L after pituitary suppression [24]. However, in the cited study, pituitary suppression was performed with depot aGnRH preceded by oral norethisterone, two hormones that are responsible for profound pituitary blockade.
In the present study, all patients in the three groups reached the final stage of follicular maturation. This was probably due to the fact that, in the control group, 13 mm follicles presented LH receptors responsive to hCG activity. Protocols that recommended the introduction of hCG after a fixed period of rFSH administration did not achieve the same success since some patients did not reach the final stages of follicular maturation [13]. Thus, our proposal of using the follicular size parameter to change the stimulation scheme and not a fixed number of days was effective, supporting the hypothesis of a more physiological role of LH in the final phase of stimulation. The growth pattern of follicles measuring <10, 10–14, and >14 mm in mean diameter along controlled ovarian stimulation and the pre-ovulatory serum concentrations of E2 and T did not differ among the three treatment groups. Similarly, there was no difference in the dose of gonadotropin needed to reach the final parameters of follicular maturation. Only one patient in each group presented ovarian hyperstimulation syndrome. Our initial hypothesis was that the number of follicles measuring less than 10 mm would be smaller in the hCG scheme, as suggested by Filicori et al. [13]. In the cited study, the effect of a low hCG dose was assessed after one week of controlled ovarian stimulation with rFSH (150 IU/day) in GnRH agonist-suppressed normoovulatory women. Four different protocols were tested: 150 IU/day of rFSH alone, 50 IU/day rFSH plus 50 IU/day hCG, 25 IU/day rFSH plus 100 IU/day hCG, and 200 IU/day hCG alone. No significant difference was detected among the four groups regarding the number of pre-ovulatory follicles measuring 10–14 and >14 mm or the pre-ovulatory concentration of estradiol. However, there was a reduction of pre-ovulatory ovarian follicles <10 mm in the patients who received hCG and the amount of rFSH necessary to reach the final parameters of maturation was progressively reduced with the increasing proportion of hCG administered. The decrease of smaller follicles was correlated with a lower chance of ovarian hyperstimulation syndrome [14]. These results indicate a combination of safety and lower costs because of the reduced dose of gonadotropin used. However, the results of the present study regarding the number of follicles <10 mm and the gonadotropin dose needed for follicular maturation differ from those reported by Filicori et al. [13]. On the other hand, our study agrees with that of Filicori et al. [13,15,16] by demonstrating that stimulation with hCG in the final follicular phase is effective and does not require exogenous FSH, with a consequent reduction of total treatment costs. Regarding the results of the assisted reproduction process, such as number of oocytes retrieved, embryos formed and transferred, and fertilization, implantation and pregnancy rates, the present series is insufficient for an analysis with a significant test power. However, it is tempting to speculate if the difference in pregnancy rate would be significant with a larger series. Similarly, in view
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of the above results, we may attribute a positive role to LH administered in the late follicular phase regarding oocyte quality. Recent studies [16,25] obtained similar results when assessing the use of selective hCG in the late follicular phase in a larger series. There are also reports of successful controlled ovarian stimulation cycles in normoovulatory women with the use of rLH alone in the late follicular phase after the initial stimulation with rFSH [26]. An interesting feature of the present study was that, despite the significantly high P levels on the pre-ovulatory hCG day in the hCG group compared to the hMG and rFSH groups, there was no deleterious effect on the final results. It has been reported that 2–35% of controlled ovarian stimulation cycles suppressed with aGnRH present serum P elevation during treatment [27]. The clinical impact of early luteinization, demonstrated by a sudden increase in P (with a cut-off level ranging from 0.5 to 2.0 ng/mL in the literature) on the day preovulatory hCG day continues to be controversial. While some studies have reported that a serum increase of this hormone during the pre-ovulatory period has a negative impact on pregnancy rates after assisted reproduction [28], others disagree and consider the difference in serum P levels on the day of pre-ovulatory hCG administration not to be correlated with clinical pregnancy rate in assisted reproduction [27,29]. In an oocyte donation program, early luteinization was associated with an increase in pregnancy rates [30]. Recently, Martinez et al. [31] demonstrated by means of the receiver–operating characteristic (ROC) curve that serum P concentration on the hCG day cannot predict the occurrence of pregnancy in assisted reproduction cycles using aGnRH and gonadotropins. These findings also permit us to propose that the pre-ovulatory elevation in P does not depend on LH. No significant differences regarding pregnancy or abortion rates were observed between groups with P levels lower or higher than 0.9 ng/mL.
5. Conclusions The present study demonstrated successful results of assisted reproduction processes with controlled ovarian stimulation protocols by using selective LH activity in the late follicular phase. With an ovarian response pattern similar to that obtained with hMG and with rFSH, it is possible to use a protocol of significantly lower cost. The protocol evaluated (hCG group) produced pregnancy rates similar to those produced in the hMG and rFSH groups even though serum P levels were significantly higher on the hCG day. Further studies conducted on larger series to assess the use of hCG in the late follicular phase are needed in order to demonstrate that efficacy and safety are comparable to those obtained in cycles supplemented with hMG in the same phase or with rFSH alone. Analysis of the oocyte quality of patients submitted to induction with hCG at the end of the follicular phase may contribute to the investigation of the role of LH during this period.
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6. Condensation Ovulation induction with hCG during the late follicular phase induces the same response pattern as that obtained with hMG or rFSH during the same phase.
Acknowledgments We wish to thank the Center of Quantitative Methods (CEMEQ) of FMRP-USP for collaboration with the statistical analysis.
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