Comparison of serum and follicular fluid hormone levels with recombinant and urinary human chorionic gonadotropin during in vitro fertilization Peter Kovacs, M.D., a Timea Kovats, M.D., a Artur Bernard, M.D., a Janos Zadori, M.D., b Gabor Szmatona, M.D., a and Steven G. Kaali, M.D. a a Kaali Institute IVF Center, Budapest, and b Kaali Institute IVF Center, Szeged, Hungary Objective: To study serum and follicular fluid (FF) hormone levels after the administration of urinary or recombinant hcg to initiate the final stages of oocyte maturation during IVF. Design: Prospective randomized study between 250 mg of recombinant hcg and 7,500 IU of urinary hcg as the final trigger of ovulation during IVF. Setting: Private IVF center. Patient(s): Infertile women undergoing IVF/intracytoplasmic sperm injection (ICSI) using the long protocol and recombinant FSH. Intervention(s): IVF treatment. Serum and FF hormone measurements on the day of oocyte collection. Main Outcome Measure(s): Serum and FF E 2, P, hcg, and T levels. Result(s): Stimulation parameters, serum and follicular E 2, P, T, and hcg levels were similar in the recombinant and urinary hcg groups. The number of oocytes retrieved from follicles >14 mm, the proportion of mature oocytes, fertilization rate, and pregnancy rate (PR) were also comparable. Conclusion(s): Recombinant and urinary hcg provided similar serum and follicular hormonal environments during the final stages of oocyte maturation. The IVF outcome parameters were also comparable. The two medications appear to be equally effective. (Fertil Steril Ò 2008;90:2133 7. Ó2008 by American Society for Reproductive Medicine.) Key Words: Recombinant, urinary, human chorionic gonadotropin, follicular fluid, steroid, serum During a spontaneous menstrual cycle a preovulatory LH surge initiates the ovulatory process. As a result of the LH surge the oocytes complete meiosis I and enter meiosis II, the cumulus oocyte complex separates from the follicle wall and the process resulting in the release of the oocyte cumulus complex is started (1). During an IVF cycle premature luteinization could negatively affect treatment outcome. If the follicles rupture prematurely, oocytes cannot be collected. To prevent premature LH surges GnRH agonist or antagonist is combined with gonadotropins. Both GnRH preparations successfully prevent premature ovulation. Therefore to collect mature eggs for fertilization the LH surge needs to be induced medically (2, 3). In cycles without down-regulation and in cycles where a GnRH antagonist is used to prevent the premature LH surge both GnRH agonist or hcg can be administered for this purpose (4, 5). In GnRH agonist down-regulated cycles hcg has to be administered before the retrieval. Human chorionic gonadotropin and LH have a lot of similarities in their structure, therefore hcg is capable of inducing the necessary changes through the LH receptor (6). Received August 22, 2007; revised and accepted October 9, 2007. Presented at the 63 rd Annual Meeting of the American Society for Reproductive Medicine, October 13 17, 2007, Washington, D.C. Reprint requests: Peter Kovacs, M.D., Kaali Institute IVF Center, Istenhegyi u 54/a, 1125 Budapest, Hungary (FAX: 36-1-214-6050; E-mail: peterkovacs1970@hotmail.com). The growing follicle requires a unique endocrine environment to develop a mature egg. Steroid hormones and various peptides produced both locally and at distant sites are responsible for providing an optimal nourishing milieu. Changes in this environment might lead to oocytes that are less likely to fertilize successfully or might result in poorer quality embryos. Previous studies have shown that an environment rich in androgens is unfavorable for the developing oocyte. Xia and Younglai (7) found that when the follicular fluid (FF) E 2 -to-t ratio exceeded 200, significantly more high quality (grades 3, 4) oocytes were obtained. Higher FF E 2 -to-t ratio was reported in IVF cycles that resulted in a pregnancy (8). Significantly higher FF P-to-E 2 ratio was measured in follicles from which the oocyte was successfully fertilized (9). Kreiner et al. (10) observed that FF E 2 and P levels tended to be within a given range in IVF cycles that resulted in a pregnancy. More recently a follicular environment richer in E 2 was reported with the use of highly purified hmg when it was compared to recombinant FSH. This group also reported that the FF E 2 -to-t ratio was higher in those follicles that provided an egg resulting in a pregnancy (11). Although the follicular environment is primarily influenced by the type of gonadotropin the follicle is exposed to during the follicular phase, the hcg injection that triggers the luteinizing changes also has a significant influence on this environment. 0015-0282/08/$34.00 Fertility and Sterility â Vol. 90, No. 6, December 2008 2133 doi:10.1016/j.fertnstert.2007.10.015 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
For decades hcg derived from urine was available and was used during IVF cycles. More recently recombinant hcg became available. Several clinical studies have shown that the two hcg preparations result in a similar number of oocytes, clinical pregnancies, ongoing pregnancies, delivery and miscarriage rates (12, 13). In our study we were interested in the follicular hormonal environment with the available urinary and recombinant hcg preparations. As secondary outcomes, we were also interested in any possible association between the hormonal values/ratios and oocyte quality, fertilization, embryo development, and pregnancy rates (PR). MATERIALS AND METHODS In this prospective, randomized multicenter trial, Institutional Review Board (IRB) approval was obtained (SZTE Albert Szent-Gyorgyi School of Medicine 106/2006) and all participants signed informed consent before entering the study. Sixty women were randomized to receive either 250 mg of recombinant hcg SC (Ovitrelle, Serono, Italy) or 7,500 IU of urinary hcg IM (Choragon, Ferring, Germany) to induce the final maturation of the oocytes. Block randomization (blocks of 2) was used to allocate patients to the two hcg preparations. Patients were randomized when they came in for the suppression check during the GnRH agonist. The study was conducted between December 2006 and April 2007. Infertile women less than 40 years of age with regular menstrual cycles were eligible to participate. The baseline FSH level had to be <12 IU/L and the uterine cavity had to be intact. Couples with severe male factor infertility (need for surgical sperm extraction or use of donor sperm) were excluded. Couples with previous poor response to stimulation (<3 oocytes, cancelled cycle, use of high dose of gonadotropins [>300 IU/day]) and those with more than two failed previous IVF cycles were also excluded. Multiple ovarian follicle development was achieved using the GnRH agonist long protocol and recombinant FSH. The GnRH agonist (0.5 ml of Suprefact SC; Aventis Pharma, Germany) was started in the luteal phase of the previous cycle. After 10 12 days of GnRH agonist administration, once suppression was confirmed, stimulation with 150 IU of recombinant FSH (Gonal-F, Serono, Italy) was started. The first ultrasound was performed on day 6 of stimulation. The dose of the gonadotropin could be increased or decreased at this point based on response. Ultrasound and, if required, serum E 2 measurements were used to monitor follicle growth. Once two leading follicles reached 17 mm in diameter recombinant or urinary hcg was administered to induce final follicular maturation. Transvaginal oocyte retrieval was scheduled 35 36 hours after this final injection. After the retrieval the oocytes were evaluated for maturity and conventional insemination or if needed intracytoplasmic sperm injection (ICSI) was used for fertilization. Fertilization was assessed the next day. The embryos were cultured for 3 5 days before the transfer. The day of transfer and the number of embryos transferred were decided by the physician based on patient and cycle characteristics. The luteal phase was supported by 600 mg of vaginal micronized P in divided doses (Utrogestan, Lab Besius, France). Twelve days after the transfer a serum b-hcg was measured. At 6 weeks and at 8 weeks a pelvic ultrasound was performed to monitor the early pregnancy. Progesterone supplementation was continued until week 9 of pregnancy. Serum and Follicular Fluid Samples On the day of the oocyte retrieval serum and FF were collected. Blood was drawn from the antecubital vein. The specimen was spun for 10 minutes at 3,000 rpm to separate the serum and cellular components. Serum measurement for E 2, P, T, and hcg were carried out with commercially available ELISA assays using an automated system. Follicular fluid was collected from several follicles. Pooled samples were obtained. All bloody samples were discarded. Follicular fluid was also spun for 10 minutes at 3,000 rpm, first to separate the cellular components. Estradiol, P, T, and hcg levels were determined using commercially available ELISA assays. For E 2 and P measurements the samples had to be diluted 1,000 times manually. Data Collection Data was collected for baseline characteristics (age, baseline FSH, E 2 ), stimulation parameters (dose of gonadotropin, type of hcg, number of follicles >14 mm, number of oocytes, rate of mature oocytes [mature MII oocytes/total oocytes retrieved], fertilization rate based on all oocytes and mature oocytes only [fertilized oocytes/all oocytes retrieved, fertilized oocytes/mature MII oocytes retrieved], number of available embryos, proportion of top quality embryos [R6 cells on day 3, <20% fragmentation; top quality embryos/all embryos], number of embryos transferred and cryopreserved), and for treatment outcome. When a positive serum pregnancy test was obtained 12 days after the embryo transfer it was considered a pregnancy. When a pregnancy progressed beyond week 8 and was discharged from our care we considered it an ongoing pregnancy. In addition to serum and FF measurements, the ratios of E 2 -to-t and E 2 -to-p were compared. Statistical Methods Student s t-test was used to compare quantitative variables and c 2 test to compare proportions between the different outcomes with recombinant and urinary hcg. A P<.05 was considered significant. RESULTS Sixty patients were randomized. Baseline characteristics were comparable in the two groups (Table 1). None of the cycles were cancelled during stimulation and all cycles ended with embryo transfer. 2134 Kovacs et al. Recombinant and urinary hcg induce similar effects Vol. 90, No. 6, December 2008
Stimulation parameters are also shown in Table 1. The stimulation parameters were similar in both groups. The follicle (>14 mm)-to-egg conversion rates were also similar with the two hcg preparations and the proportion of mature oocytes and fertilization rates were also comparable. Serum and FF hormone values in the two groups are shown in Table 2. Estradiol, P, T, and hcg levels were similar with the two hcg preparations, both in the serum and FF. The overall PR was 51.7 % (31/60). There was no significant difference in the PR (recombinant hcg: 13/30 [43.3%] vs. urinary hcg: 18/30 [60%]; P ¼.301), ongoing PR (recombinant hcg: 11/30 [36.6%] vs. urinary hcg: 15/30 [50%]; P ¼.73), and multiple gestation rate (recombinant hcg: 2/13 [15.3%] vs. urinary hcg: 6/18 [33.3%]; P ¼.395) in the two groups. DISCUSSION Normal follicle development requires a very specific microenvironment. Although the final stages of folliculogenesis primarily depend on the availability of FSH and LH, various growth factors, peptide hormones, and cytokines contribute to this process. The last step before ovulation is the LH surge. The surge is needed for the completion of meiosis I, for the release of the oocyte, and also for the transition to corpus luteum (CL) function. As a result of the LH surge E 2 production is decreased in the granulosa cells (GC) and P synthesis increases at the expense of androgen synthesis in the theca cells (1). During an IVF cycle the spontaneous LH surge is blocked and when needed the LH surge is induced with either GnRH agonist or hcg (4, 5). The hcg has a similar structure to LH and is capable of binding to the LH receptor and to mediate an effect through it (6). For a long time hcg was only available after purification from urine. More recently a recombinant form became available. Several studies have compared their clinical effects. Driscoll et al. (14), in a randomized study, found that the number of oocytes collected per follicles aspirated, number of mature oocytes, fertilized oocytes, and number of cleaving embryos were similar with 5,000 IU of urinary hcg or 250 mg of recombinant hcg. More recently several meta-analyses and review articles confirmed similar effects (12, 13). It has been previously reported that the steroid and peptide microenvironment within the follicle reflects on the competence of the egg retrieved from that follicle. It was shown that those IVF cycles where the FF E 2 and E 2 -to-p ratio was in a given range were more likely to result in pregnancy (10). Other studies have also observed a correlation between steroid hormone ratios and fertilization rates during IVF cycles (9). Andersen (8) studied the association between steroid and peptide hormone levels in FF, and oocyte maturity, fertilization, embryo cleavage, and pregnancy outcome. He also demonstrated in his study that rather than individual steroid levels, the FF E 2 -to-t ratio was the best predictor of successful implantation. Higher ratio (suggesting better androgen-to-estrogen [E] conversion) was associated with higher implantation rate (8). Another group studied the correlation between oocyte quality (assessed by the status of the TABLE 1 Demographic and stimulation characteristics in the recombinant and urinary hcg groups. Recombinant hcg group Urinary hcg group Age (y) 31 0.6 32.1 0.6 Baseline FSH (IU/L) 6.7 0.3 7.2 0.3 Baseline E 2 (pmol/l) 138.8 9.1 122.3 12.1 Gonadotropin (IU) 1342.6 77.7 1423.6 78.0 Follicles >14 mm 8.5 0.5 9.0 0.6 Oocytes 9.3 0.7 10.3 1.0 MII oocytes 7.0 0.6 8.2 0.9 Rate of mature MII oocytes (%) 80.6 8.5 76.7 3.5 Fertilized oocytes 5.1 0.4 6.3 0.8 Fertilization rate per all oocytes (%) 61.2 8.0 58.9 4.7 Fertilization rate per mature (MII) oocytes (%) 73.2 5.2 76.7 5.0 No. of embryos 4.8 0.4 6.1 0.8 No. of top quality embryos 2.3 0.3 3.2 0.4 Rate of top quality embryos (%) 49.5 4.7 55.6 5.0 No. of embryos transferred 2.2 0.1 2.1 0.1 No. of embryos frozen 0.6 0.2 1.1 0.5 Note: Values are expressed as mean SEM. Using t test for comparison at P<.05 none of the differences are significant. Kovacs. Recombinant and urinary hcg induce similar effects. Fertil Steril 2008. Fertility and Sterility â 2135
TABLE 2 Serum and follicular fluid hormone levels on the day of the retrieval in the two experimental groups. Recombinant hcg group Urinary hcg group Serum E 2 (pmol/l) 4,934.9 813.9 4,058.8 597.2 P (nmol/l) 47.5 14.0 34.0 9.9 hcg (IU/L) 141.9 9.2 161.6 12.4 T (nmol/l) 3.4 0.2 3.0 0.2 Follicular fluid E 2 (pmol/l) 2,717,152 336,649 2,154,740 262,745 P (nmol/l) 35,273 5,076 37,371 5,080 hcg (IU/L) 113.8 31.4 119.6 28.8 T (nmol/l) 23.8 2.6 24.8 3.8 E 2 :T 171,647.6 53,645.1 192,032.0 68,716.5 E 2 :P 135.4 24.2 90.7 16.8 Note: Values are expressed as mean SEM. Using t test for comparison at P<.05 none of the differences are significant. Kovacs. Recombinant and urinary hcg induce similar effects. Fertil Steril 2008. polar body and the size of the perivitelline space) and follicular steroid hormone levels. They measured higher E 2 -to-t ratio in those follicles that contained higher quality oocytes (7). Tessier et al. (15) showed that individual steroid levels also differed between meiotically competent and incompetent oocytes. The concentration of E 2 and P was significantly higher in follicles that contained meiotically competent oocytes, whereas the concentration of T was higher in follicles that contained meiotically incompetent oocytes in both normal and women with PCOS. The purpose of our study was to evaluate whether urinary and recombinant hcg were equally capable of initiating the conversion of follicular activity to CL activity and thereby whether they are equally effective in providing the necessary hormonal environment for the oocytes before retrieval. We did not evaluate individual follicles but were interested in the overall performance of the two drugs, therefore we used pooled samples. Patients with similar baseline characteristics, treated with the same stimulation protocol and similar amount of gonadotropin were analyzed. 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