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Does prolonged pituitary down-regulation with gonadotropin-releasing hormone agonist improve the live-birth rate in in vitro fertilization treatment?
ORIGINAL ARTICLE: ASSISTED REPRODUCTION
Does prolonged pituitary down-regulation with gonadotropin-releasing hormone agonist improve the live-birth rate in in vitro fertilization treatment? Jianzhi Ren, M.D., Aiguo Sha, M.D., Dongmei Han, M.S., Ping Li, M.S., Jie Geng, M.S., and Chaihui Ma, M.S. Reproductive Medicine Center, Affiliated Chenggong Hospital of Xiamen University, Xiamen, Fujian, People's Republic of China
Objective: To evaluate the effects of a prolonged duration of gonadotropin-releasing hormone agonist (GnRH-a) in pituitary downregulation for controlled ovarian hyperstimulation (COH) on the live-birth rate in nonendometriotic women undergoing in vitro fertilization and embryo transfer (IVF-ET). Design: Retrospective cohort study. Setting: University-affiliated hospital. Patient(s): Normogonadotropic women undergoing IVF. Intervention(s): Three hundred seventy-eight patients receiving a prolonged pituitary down-regulation with GnRH-a before ovarian stimulation and 422 patients receiving a GnRH-a long protocol. Main Outcome Measure(s): Live-birth rate per fresh ET. Result(s): In comparison with the long protocol, the prolonged down-regulation protocol required a higher total dose of gonadotropins. A lower serum luteinizing hormone (LH) level on the starting day of gonadotropin and the day of human chorionic gonadotropin (hCG) and a fewer number of oocytes and embryos were observed in the prolonged down-regulation protocol. However, the duration of stimulation and number of high-quality embryos were comparable between the two groups. A statistically significantly higher implantation rate (50.27% vs. 39.69%), clinical pregnancy rate (64.02% vs. 56.87%) and live-birth rate per fresh transfer cycle (55.56% vs. 45.73%) were observed in the prolonged protocol. Conclusion(s): Prolonged down-regulation in a GnRH-a protocol might increase the live-birth rates in normogonadotropic women. (Fertil SterilÒ 2014;-:-–-. Ó2014 by American Use your smartphone Society for Reproductive Medicine.) to scan this QR code Key Words: Controlled ovarian hyperstimulation, gonadotropin releasing hormone agonist and connect to the (GnRH-a), in vitro fertilization Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/renj-pituitary-down-regulation-gnrha-live-birth-rate-ivf-treatment/
G
onadotropin-releasing hormone agonists (GnRH-a) are the most commonly used adjuvants for controlled ovarian stimulation
(COH). The benefits of using GnRH-a during COH in assisted reproductive treatment cycles is well documented, such as prevention of a premature lutei-
Received November 25, 2013; revised and accepted March 14, 2014. J.R. has nothing to disclose. A.S. has nothing to disclose. D.H. has nothing to disclose. P.L. has nothing to disclose. J.G. has nothing to disclose. C.M. has nothing to disclose. Supported in part by the major science and technology platform in Xiamen City (No. 3502z20111006). Reprint requests: Jianzhi Ren, M.D., Reproductive Medicine Center, Affiliated Chenggong Hospital of Xiamen University, 94 Wenyuan Road, Xiamen, Fujian 361002, People's Republic of China (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.03.030 VOL. - NO. - / - 2014
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nizing hormone (LH) surge, luteinization, a lower cycle cancellation rate (1), and the recovery of a larger number of oocytes (2). Among the various types of GnRH-a protocols, the gold standard for ovarian stimulation in young normogonadotropic women is recognized as the long protocol, starting GnRH-a in the midluteal phase of the preceding cycle (3). Down-regulation with GnRH-a is also a recommended medical pretreatment before in vitro fertilization (IVF) 1
ORIGINAL ARTICLE: ASSISTED REPRODUCTION in patients with endometriosis (4). The clinical pregnancy rates were improved when a 2- to 6-month period of hormone suppression with GnRH-a was administered. Given that women with endometriosis may have a lower pregnancy rate in comparison with women with tubal factor infertility (5), some of the reported pregnancy rates in patients pretreated with GnRH-a are rather high (6–8). In a ultralong protocol in which GnRH-a was administered for at least 60 days before ovarian stimulation, a pregnancy rate of 67% was reported (6). In their classic paper, Surrey et al. (7) reported remarkable ongoing pregnancy rates in patients who received a 3-month long-acting GnRH-a regimen when comparing them with those who received conventional midluteal phase GnRH-a down-regulation (80% vs. 53.85%). A similar result was also observed among patients who received treatment with GnRH-a for 6 months before assisted reproduction (8). These facts suggest that prolonged downregulation before IVF treatment might provide superior outcomes in patients with endometriosis. Over two decades ago, it was noted that pregnancy rates might be improved when women are given a period of amenorrhea before embryo transfer (9). High pregnancy rates were found in acyclic or postmenopausal women undergoing oocyte donation and IVF treatment in comparison with the rates among cyclic women. The age of the oocyte donor, the in vitro growth of the embryo, the proportion of defective embryos in culture, and the type of cycle during therapy were found to make no contribution to the difference (10). Edwards suggested that the high pregnancy rates were primarily caused by the previous amenorrhoea in the acyclic or postmenopausal women before the IVF treatment, rather than the use of donor oocytes. Amenorrhea induced by pituitary down-regulation with GnRH-a might also have the same effects. In a study on women undergoing IVF and hormone replacement therapy, cyclic women of their late 40s who were down-regulated for 3 months using GnRH-a were compared with acyclic women and cyclic women of similar age who did not receive GnRH-a pretreatment. A similar pregnancy rate of 30% was found in GnRH-a pretreated women and acyclic women, which was much higher than that of cyclic women without GnRH-a pretreatment (11). The early study suggested that a period of down-regulation might be a favorable factor for infertile women undergoing IVF and that this effect might due to increased endometrial receptivity. If the hypothesis is true, the general IVF population may also benefit from prolonged down-regulation before COH and embryo transfer (ET). However, down-regulation for several months before IVF-ET might not be acceptable in general patient cohort. In this study, we determined the effect of a modified long protocol with 28 days of GnRH-a down-regulation on the live-birth rate in fresh ET cycles in comparison with the long protocol.
MATERIALS AND METHODS Study Participants Institutional review board approval was obtained for this retrospective study. The retrospective analysis was performed on fresh ET cycles stimulated with agonist long protocol in 2
our center between June 2010 and December 2011. All patients had undergone a full set of infertility evaluation and were diagnosed as tubal factor infertility. Presence or absence of endometriosis was confirmed by laparoscopy, and endometritis cases were also excluded from the study. The patients whose male counterpart suffered from male infertility and required intracytoplasmic sperm injection (ICSI) treatment were also excluded. Patients were selected if they met all the following inclusion criteria: women with good physical and mental health, aged <35 years; regular menstrual cycles ranging from 25 to 35 days; body mass index (BMI) <28 kg/ m2; normal basal serum follicle-stimulating hormone (FSH) (%12 IU/L) and estradiol (E2) (%75 pg/mL) levels determined on day 3 of the cycle previous to COH; no uterine abnormalities (fibroids, adenomyosis, m€ ullerian malformations) were assessed by vaginal ultrasound. Patients had not received any ovulation-induction drug within 1 month before COH. The enrollment of patients for each protocol was simultaneous. The treatment assignment was primary decided by the physicians. Before a cycle was initiated, the patient was informed of detailed information of both protocols, including the duration of the down-regulation, the pregnancy rate in previous cycles, and the potential risk of strong pituitary suppression. Based on this information, patients made the decision to accept or reject a specific protocol. All the patients enrolled gave written informed consent for the procedures. The study criteria was met by 955 initiated cycles. Eight initiated cycles were canceled before oocyte retrieval (four in the prolonged protocol, and four in the long protocol). Thirteen cycles in the prolonged protocol group and eight cycles in long protocol group were canceled because no embryo was available. In the prolonged protocol group, 65 cycles and 61 cycles in long protocol were canceled for fresh ET because all embryos were cryopreserved. Because prolonged down-regulation is supposed to affect the endometrial receptivity, and live-birth rates are the main outcome measure in our present study, all cycles canceled for fresh ET were excluded. Therefore, 378 patients receiving the prolonged pituitary down-regulation with GnRH-a before ovarian stimulation and 422 patients receiving the GnRH-a long protocol were included (Fig. 1). The total cancellation rates of ET in the two cohorts were comparable (17.8% in prolonged protocol vs. 14.7% in long protocol; P¼ .197).
Treatment Protocol In the GnRH-a prolonged protocol (prolonged protocol), pituitary down-regulation was obtained with standard full dose (3.75 mg) of triptorelin depot (Decapeptyl 3.75; Ipsen Pharma) in the early follicular phase. The size and number of the antral follicles were monitored from day 1 or 2 of the menstrual cycle. A single intramuscular injection of GnRH-a depot was given when the diameter of the antral follicles was <6 mm. Down-regulation was confirmed after 28 days (no ovarian cysts >8 mm; E2 <50 pg/L) before the gonadotropin stimulation started. No further GnRH analogue treatment was given during the stimulation. It was previously demonstrated elsewhere that LH suppression is maintained until week 8 after the injection of a single depot of triptorelin (12). VOL. - NO. - / - 2014
Fertility and Sterility®
FIGURE 1
mean diameter. We then injected 5,000–10,000 IU of human chorionic gonadotropin (hCG; Lizhu Pharma) intramuscularly. Oocyte retrieval was scheduled for 34 to 36 hours after hCG administration. Oocyte retrieval was performed using a transvaginal ultrasound approach. Semen was produced by masturbation, and motile spermatozoa were prepared by the swim-up procedure. Oocytes were inseminated 4 to 5 hours after collection, pronuclei were identified 17 to 18 hours later, and ET was performed 68 to 70 hours after insemination. Luteal phase support was sustained with natural progesterone in oil (progesterone; XianJu), 60 mg injected intramuscularly each day from the day of oocyte retrieval. A pregnancy test (serum b-hCG determination) was performed 14 days after ET. Clinical pregnancy was defined as the presence of one or more gestational sacs detected on ultrasound scan performed 4 weeks after ET.
Statistical Analysis Analysis was performed with SPSS (version 19; IBM). Independent samples t-test or the Mann-Whitney test was used for continuous variables. Dichotomous variables were analyzed by chi-square test or Fisher's exact test, as appropriate. A multivariable logistic regression model was used to evaluate the contribution of prolonged down-regulation to the difference in live-birth rates. In all analyses, P< .05 was considered statistically significant.
RESULTS Flow sheet describing dropout of enrolled patients. Ren. Prolonged GnRH-a regimen improves outcome. Fertil Steril 2014.
In the GnRH-a long protocol (long protocol), one-third dose (1.25 mg) of triptorelin depot in a single intramuscular injection in the midluteal phase (day 21) of the menstrual cycle preceding treatment was used for pituitary downregulation. Down-regulation was confirmed after 14 days following the same criteria described in the prolonged protocol and was followed by gonadotropin stimulation. In all treatment protocols, patients received two to three ampules (150–225 IU) of gonadotropin per day during the gonadotropin stimulation. A starting dose of 150 IU was given to patients with a high antral follicular count (AFC) (>10) and low body weight (<60 kg). Recombinant FSH (Gonal F; Merck Serono) or domestic urinary human menopausal gonadotropin (hMG; Lizhu Pharma) was used for the gonadotropin stimulation. The serum LH level was determined with the use of a commercial enzyme immunoassay kit before the beginning of ovarian stimulation. When the LH level was <1 IU/L, the patients were stimulated with hMG alone, and otherwise with FSH alone. We adjusted the initial and ongoing dosage according to the patient's age, AFC, BMI, and follicular growth response. During the treatment, ovarian response was monitored by measurements of follicular growth via transvaginal ultrasound and serum E2 levels every 1 to 3 days. Gonadotropin stimulation continued until ultrasonography revealed at least one follicle measuring R18 mm in VOL. - NO. - / - 2014
The average age of the 800 women enrolled in the present study was 30.37 3.49 years (mean standard deviation [SD]), and the mean duration of infertility was 4.06 3.79 years (mean SD). No statistically significant difference was noted in age, infertile period, or BMI in comparing the two groups (Table 1). The basal serum hormone levels were also comparable between the patient cohorts, except for the FSH level. Patients receiving prolonged protocol showed a statistically significantly higher basal FSH level. A statistically significant difference was found in the total doses taken for ovarian stimulation (P¼ .0001); more doses were needed for hyperstimulation in the prolonged protocol group than in the long protocol group, although there was no statistically significant difference in the total days taken for ovarian stimulation (P¼ .235). On the first day of gonadotropin stimulation, the serum LH and E2 level was lower in the prolonged protocol group, which suggested a stronger suppression on pituitary and ovary. On the day of hCG, the serum LH level of prolonged protocol remained lower. However, in terms of the serum level of E2 and P, there were no differences between the two groups. The numbers of AFC on first day of stimulation, oocytes retrieved, fertilized, cleaved, clinical pregnancy, implantation rate, early miscarriage rate, and live-birth rate in both treatment protocols are given. There were greater AFC, oocytes retrieved, and fertilized and cleaved eggs in the long protocol group, but the total numbers of high-quality embryos were similar in both groups. Endometrial thickness on the day of hCG administration was significantly thicker in the prolonged protocol group. There was no difference in the number of 3
ORIGINAL ARTICLE: ASSISTED REPRODUCTION
TABLE 1 Basic characteristics and stimulation variables of the two groups.
Patient characteristics Age (y) Duration of infertility (y) Body mass index Stimulation characteristics Length of stimulation (d) Gonadotropin units (IU) FSH on day 3 (mIU/mL) LH on day 3 (mIU/mL) E2 on day 3 (pg/mL) Antral follicular count on 1st day of stimulation FSH on day 1 of stimulation (mIU/mL) LH on day 1 of stimulation (mIU/mL) E2 on day 1 of stimulation (pg/mL) E2 on day of hCG (pg/mL) LH on day of hCG (mIU/mL) Progesterone on day of hCG (ng/mL) Endometrial thickness on day of hCG (mm) No. of oocytes retrieved No. of oocytes fertilized No. of oocytes cleaved No. of high-quality embryos No. of embryos transferred Cycle outcomes Clinical pregnancy per ET (%) Implantation rate Early abortion rate (%) Live birth per ET (%)
Prolonged (n [ 378)
Long (n [ 422)
P value
29.44 2.82 3.5 [2–5] 20.94 2.60
29.22 3.05 3 [2–5] 21.04 2.58
.315 .763 .446
10.53 1.60 29.61 6.21 6.36 1.94 4.32 2.02 36.11 19.78 8.21 3.44 2.47 1.27 0.85 0.44 12.43 9.35 2,654.76 1,318.26 0.85 0.80 0.92 0.54 1.11 0.23 9.61 4.21 7.45 3.91 5.85 3.23 3 [1–5] 1.95 0.38
10.59 1.82 26.40 6.97 5.45 1.40 4.79 3.02 37.66 27.39 9.95 2.98 1.93 1.21 1.24 0.67 13.29 6.80 2,760.88 1,213.30 1.22 1.00 0.85 0.47 1.06 0.18 10.73 4.01 8.23 3.62 6.66 3.11 3 [2–6] 1.98 0.26
.805 .001 .001 .354 .777 .001 .001 .001 .001 .133 .000 .136 .017 .001 .002 .001 .074 .264
242/378 (64.02) 50.27% 22/242 (9.09) 210/378 (55.56)
240/422 (56.87) 39.69% 31/240 (12.92) 193/422 (45.73)
.039 .001 .179 .006
Note: Numbers are mean þ standard deviation (range) or median [interquartile range]. E2 ¼ estradiol; ET ¼ embryo transfer; FSH ¼ follicle-stimulating hormone; hCG ¼ human chorionic gonadotropin; LH ¼ luteinizing hormone. Ren. Prolonged GnRH-a regimen improves outcome. Fertil Steril 2014.
embryos transferred. There were higher rates of clinical pregnancy (64.02% vs. 56.87%, P¼ .039) and implantation (50.27% vs. 39.69%, P< .001) in the prolonged protocol group. There was no statistically significant difference between the groups in the spontaneous abortion rate. The live-birth rate in the prolonged protocol group was statistically significantly increased (55.56% vs. 45.73%, P¼ .006). To evaluate the contribution of the period of downregulation to the difference in live-birth rate, a multivariate logistic regression model was used. The variables that were different between the two patient cohorts (basal FSH levels, AFC, FSH, LH, and E2 levels on the day of stimulation, LH levels and endometrial thickness on the day of hCG, number of oocytes retrieved) were selected as potential confounding factors. Other factors (age, progesterone elevation on the hCG day, number of good quality embryo) were also selected based on clinical experiences, although they were similar in both cohorts. The Hosmer-Lemshow goodness-of-fit chi-square test statistic was 8.75 (P¼ .364), which suggested that the multivariable model was a good fit (P>.05). Adjusted by the previously mentioned potential confounding factors, the odds ratio of prolonged down-regulation versus 14-day down-regulation on the live-birth rate was 1.464 (95% confidence interval, 1.069–2.131).
DISCUSSION Several studies using prolonged GnRH-a pretreatment before IVF have obtained relatively high pregnancy rates (6–8). 4
However, in these studies the sample sizes of each group were small, and the pathology of endometriosis was a major confounding factor. In a randomized controlled trial involving 60 patients, Fabregues et al. (13) reported that monthly prolonged down-regulation with GnRH-a did not improve outcomes in women who did not have endometriosis. Their sample size calculation was based on the assumption that a 30% difference in favor of long-term down-regulation versus controls existed, which was obtained from a study with women aged in their late 40s. For normogonadotropic women of reproductive age, it is unlikely that a 30% improvement in pregnancy rate can be achieved solely by long-term downregulation. To detect or reject the existence of a smaller difference might require a larger sample size. Our data argue that prolonged down-regulation still has a beneficial effect on the live-birth rate. The effect of prolonged down-regulation also has been observed in women who do not achieve timely suppression with daily dosed GnRH-a. Loutradis et al. (14) compared the IVF outcomes of prolonged GnRH-a in a small group of patients whose administration period was shorter or longer than 15 days, and found a favorable effect of GnRH-a prolongation on embryo cleavage speed and pregnancy rate. Later, these investigators reported that no difference in cycle outcome or clinical pregnancy was found between women receiving a 14- to 24-days' or 25- to 40-days' desensitization (15). More recently, Dessolle et al. (16) reported similar livebirth rates per transfer (28.86% vs. 32%) in women receiving VOL. - NO. - / - 2014
Fertility and Sterility® 14-days of down-regulation and in women who needed 7 to 28 additional days to achieve complete suppression. These studies suggested that prolonging GnRH-a might not compromise the outcomes even if it did not provide a favorable effect. However, it is worth bearing in mind that these reports were based on patient cohorts who had failed to achieve satisfactory suppression in a standard-duration down-regulation protocol; why they required a longer GnRH-a course is not fully understood. Our data showed that in the cohort with prolonged downregulation, the endometrial thickness was statistically significantly higher. The thickness of the endometrium has been used as a marker of adequate receptivity of the uterus and as a prognostic factor in ET. Richter et al. (17) demonstrated a significant increase in the pregnancy rate as the endometrial thickness increased, independent of the number or quality of the embryos transferred. Their conclusion was confirmed by Al-Ghamdi et al. (18) in a 2,464-cycle cohort study. More recently, Chen et al. (19) reported results that were closely similar to those two studies. It is well known that endometrial growth and differentiation are sensitive to steroid hormones; Edwards (9) hypothesized that steroid-responsive systems in the uterus become impaired after years of regular menstrual cycles, so a period of amenorrhea or down-regulation might recover the systems from this constant stimulus. A greater thickness of endometrium achieved by prolonged downregulation might support the hypothesis of endometrial recovery. Prolongation of down-regulation also results in enhanced exposure to GnRH-a. The GnRH-a may contribute to the outcomes by an effect on the embryo or a direct effect on the endometrium. The assessment of embryo quality is difficult to establish, but after long suppression of the pituitary-ovarian axis, the function of oocytes or intrafollicular dynamics might be induced and improve embryo quality. A study in an animal model suggested that preimplantation embryonic development was significantly enhanced by incubation with increasing concentrations of GnRH-a, and noted that the expression of GnRH and its receptor were also observed in human preimplantation embryos (20). Previous studies suggested that GnRH-a treatment may have a direct effect on endometrium by regulating the expression of the enzymes and cytokines (21–23). Intracellular signals of the transforming growth factor-b pathway, interleukin-1b and vascular endothelial growth factor production, and extracellular-signal-related kinase activation in human endometrial cells were all found to be affected by GnRH-a binding (24, 25). In mice, the expression of endometrial receptivity markers such as integrin-b3 subunit and leukemia inhibitory factor (LIF) is increased in ovarian stimulation with GnRH-a (26). However, direct evidence supporting the role of GnRH-a in human embryo implantation remains limited. Schachter et al. (27) reported in a randomized study that administering GnRH-a before oocyte retrieval in GnRH antagonist cycles may improve pregnancy rates. In an oocyte donation program, after a single dose of 0.1 mg triptorelin administered 3 days after ET (28), a higher implantation rate was obtained compared with the control group who received VOL. - NO. - / - 2014
placebo at the same time. The same investigators later reported similar results in both GnRH-a- and antagonisttreated ovarian stimulation cycles (29). Studies with frozen embryo transfer (FET) cycles provide an opportunity to estimate the effects of GnRH-a on oocyte and endometrium separately. It suggests that the embryos derived from GnRH-a cycles might not be superior to those from GnRH antagonist cycles, as the outcomes in subsequent FET cycles are comparable (30–33). In contrast, administering GnRH-a before ET in artificial FET cycles might improve pregnancy rates in comparison with natural cycles (34) or artificial cycles without GnRH-a (35). Although the role of GnRH-a in endometrium preparation remains controversial (36), the collective findings in fresh and FET cycles suggest a role for GnRH-a in improving embryo implantation. In our study, a full-dose depot GnRH-a injection was used to achieve suppression in the prolonged protocol. Although the formulation was primarily used to provide pituitary suppression for 28 days, a previous study demonstrated that a full-dose depot GnRH-a injection was sufficient to maintain LH suppression until week 8 after the injection, and estrogen secretion started to be restored in the course of weeks 7–8. Suppression of pituitary and ovarian function appears to be continued until week 8 after the injection (12). In line with previous findings, our data showed that an LH surge was prevented until the day of oocyte retrieval and that no additional GnRH analogues were needed during ovarian stimulation. However, full-depot GnRH in the long protocol was also associated with strong pituitary suppression (37), which might impair the ovarian response to exogenous FSH. Studies have shown that a half-dose (37) or one-third dose (38) depot triptorelin injection was sufficient to prevent LH surges and to improve the ovarian response. In comparison with those down-regulated with one-third dose depot triptorelin for the long protocol, the prolonged protocol required more exogenous gonadotropin and yielded fewer oocytes. However, the number of good quality embryos was unaffected, and the live-birth rate was significantly higher. The benefit of prolonged down-regulation might have partially balanced the effect of the drawback of strong suppression. However, in our present study, the participants were limited to young, normogonadotropic women. For patients who have responded poorly in previous cycles or patients with predictors of poor response, loss of ovarian response after prolonged downregulation could become a significant issue. In which case, alternative protocols should be taken into consideration. A reduced LH level after strong pituitary suppression may be detrimental to oocyte quality and uterine receptivity (39). When LH levels were <0.5 IU/L, this led to significantly lower E2 production, fewer oocytes, a lower fertilization rate, and fewer embryos available for cryopreservation (40–42). An increased risk of early pregnancy loss has also been suggested (42). The work of Dal Prato et al. (37) has suggested that low LH levels associated with full-dose depot GnRH-a in a long protocol may affect the clinical outcomes in patients stimulated with recombinant FSH. In contrast, a similar randomized trial in which patients were stimulated with hMG failed to find the same association (43). The difference between the results of the two studies probably lies in the 5
ORIGINAL ARTICLE: ASSISTED REPRODUCTION gonadotropin used. Because hMG contains nearly equal activity of FSH and LH, it might contribute as a source of exogenous LH supplementation. The addition of exogenous LH in patients whose serum LH has been suppressed might increase the number of developmentally competent oocytes as well as the rate of implantation (39). In our present study, patients with a serum LH of <1 IU/l might also have benefited from hMG stimulation. The nonrandomized design is a significant weakness in our study. Although a nonrandomized trial has its own advantages, it is always associated with selection bias issues. With selection bias being introduced, one might expect the result will be skewed in some ways. For example, patients with good ovarian response might be more likely to be assigned to the prolonged group, in which the suppression is stronger. However, a higher basal FSH level in prolonged protocol might suggest the opposite. Therefore, adjusting for covariates is important for interpreting the results. Methodologic study suggests that adjusted results from nonrandomized studies with a rich set of covariates can approximate results from randomized experiments (44). Although the unknown and unmeasured confounders remained to be unadjusted for in our study, the results establish a crude baseline that can be compared with the results of subsequent studies. In conclusion, our data suggest that a prolonged ovarian suppression of 28 days with a standard full dose before ovarian stimulation in IVF-ET might improve the live-birth rate per fresh ET. The effects might be associated with increased endometrial receptivity during embryo implantation, although the precise mechanism remains unclear. The study is limited by its retrospective nature as well as the specified patient cohort. The patient cohort consisted of young, normogonadotropic women. Based on previous reports (9), a more profound effect on endometrium might be expected in patients of advanced age. However, a decrease in ovarian reserve and oocyte quality are also age related. Whether prolonged down-regulation for 28 days can improve the livebirth rate among women of advanced age is unknown. Acknowledgments: The authors thank all the staff, nurses, and physicians at the Reproductive Medicine Center for their support in generating this manuscript.
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Meresman GF, Bilotas MA, Lombardi E, Tesone M, Sueldo C, Baranao RI. Effect of GnRH analogues on apoptosis and release of interleukin-1beta and vascular endothelial growth factor in endometrial cell cultures from patients with endometriosis. Hum Reprod 2003;18:1767–71. Ruan HC, Zhu XM, Luo Q, Liu AX, Qian YL, Zhou CY, et al. Ovarian stimulation with GnRH agonist, but not GnRH antagonist, partially restores the expression of endometrial integrin b3 and leukaemia-inhibitory factor and improves uterine receptivity in mice. Hum Reprod 2006;21:2521–9. Schachter M, Friedler S, Ron-El R, Zimmerman AL, Strassburger D, Bern O, et al. Can pregnancy rate be improved in gonadotropin-releasing hormone (GnRH) antagonist cycles by administering GnRH agonist before oocyte retrieval? A prospective, randomized study. Fertil Steril 2008;90:1087–93. Tesarik J, Hazout A, Mendoza C. Enhancement of embryo developmental potential by a single administration of GnRH agonist at the time of implantation. Hum Reprod 2004;19:1176–80. Tesarik J, Hazout A, Mendoza-Tesarik R, Mendoza N, Mendoza C. Beneficial effect of luteal-phase GnRH agonist administration on embryo implantation after ICSI in both GnRH agonist- and antagonist-treated ovarian stimulation cycles. Hum Reprod 2006;21:2572–9. Bahceci M, Ulug U, Erden HF, Tosun S, Ciray N. Frozen-thawed cleavagestage embryo transfer cycles after previous GnRH agonist or antagonist stimulation. Reprod Biomed Online 2009;18:67–72. Lee JR, Choi YS, Jee BC, Ku SY, Suh CS, Kim KC, et al. Cryopreserved blastocyst transfer: impact of gonadotropin-releasing hormone agonist versus antagonist in the previous oocyte retrieval cycles. Fertil Steril 2007;88: 1344–9. Eldar-Geva T, Zylber-Haran E, Babayof R, Halevy-Shalem T, Ben-Chetrit A, Tsafrir A, et al. Similar outcome for cryopreserved embryo transfer following GnRH-antagonist/GnRH-agonist, GnRH-antagonist/HCG or long protocol ovarian stimulation. Reprod Biomed Online 2007;14:148–54. Medved R, Virant-Klun I, Meden-Vrtovec H, Tomazevic T. Outcome of frozen-thawed blastocysts derived from gonadotropin releasing hormone agonist or antagonist cycles. J Assist Reprod Genet 2006;23:275–9. Hill MJ, Miller KA, Frattarelli JL. A GnRH agonist and exogenous hormone stimulation protocol has a higher live-birth rate than a natural endogenous hormone protocol for frozen-thawed blastocyst-stage embryo transfer cycles: an analysis of 1391 cycles. Fertil Steril 2010;93:416–22.
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7
Does prolonged pituitary down-regulation with gonadotropin-releasing hormone agonist improve the live-birth rate in in vitro fertilization treatment? Jianzhi Ren, M.D., Aiguo Sha, M.D., Dongmei Han, M.S., Ping Li, M.S., Jie Geng, M.S., and Chaihui Ma, M.S. Reproductive Medicine Center, Affiliated Chenggong Hospital of Xiamen University, Xiamen, Fujian, People's Republic of China
Objective: To evaluate the effects of a prolonged duration of gonadotropin-releasing hormone agonist (GnRH-a) in pituitary downregulation for controlled ovarian hyperstimulation (COH) on the live-birth rate in nonendometriotic women undergoing in vitro fertilization and embryo transfer (IVF-ET). Design: Retrospective cohort study. Setting: University-affiliated hospital. Patient(s): Normogonadotropic women undergoing IVF. Intervention(s): Three hundred seventy-eight patients receiving a prolonged pituitary down-regulation with GnRH-a before ovarian stimulation and 422 patients receiving a GnRH-a long protocol. Main Outcome Measure(s): Live-birth rate per fresh ET. Result(s): In comparison with the long protocol, the prolonged down-regulation protocol required a higher total dose of gonadotropins. A lower serum luteinizing hormone (LH) level on the starting day of gonadotropin and the day of human chorionic gonadotropin (hCG) and a fewer number of oocytes and embryos were observed in the prolonged down-regulation protocol. However, the duration of stimulation and number of high-quality embryos were comparable between the two groups. A statistically significantly higher implantation rate (50.27% vs. 39.69%), clinical pregnancy rate (64.02% vs. 56.87%) and live-birth rate per fresh transfer cycle (55.56% vs. 45.73%) were observed in the prolonged protocol. Conclusion(s): Prolonged down-regulation in a GnRH-a protocol might increase the live-birth rates in normogonadotropic women. (Fertil SterilÒ 2014;-:-–-. Ó2014 by American Use your smartphone Society for Reproductive Medicine.) to scan this QR code Key Words: Controlled ovarian hyperstimulation, gonadotropin releasing hormone agonist and connect to the (GnRH-a), in vitro fertilization Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/renj-pituitary-down-regulation-gnrha-live-birth-rate-ivf-treatment/
G
onadotropin-releasing hormone agonists (GnRH-a) are the most commonly used adjuvants for controlled ovarian stimulation
(COH). The benefits of using GnRH-a during COH in assisted reproductive treatment cycles is well documented, such as prevention of a premature lutei-
Received November 25, 2013; revised and accepted March 14, 2014. J.R. has nothing to disclose. A.S. has nothing to disclose. D.H. has nothing to disclose. P.L. has nothing to disclose. J.G. has nothing to disclose. C.M. has nothing to disclose. Supported in part by the major science and technology platform in Xiamen City (No. 3502z20111006). Reprint requests: Jianzhi Ren, M.D., Reproductive Medicine Center, Affiliated Chenggong Hospital of Xiamen University, 94 Wenyuan Road, Xiamen, Fujian 361002, People's Republic of China (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.03.030 VOL. - NO. - / - 2014
discussion forum for this article now.*
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nizing hormone (LH) surge, luteinization, a lower cycle cancellation rate (1), and the recovery of a larger number of oocytes (2). Among the various types of GnRH-a protocols, the gold standard for ovarian stimulation in young normogonadotropic women is recognized as the long protocol, starting GnRH-a in the midluteal phase of the preceding cycle (3). Down-regulation with GnRH-a is also a recommended medical pretreatment before in vitro fertilization (IVF) 1
ORIGINAL ARTICLE: ASSISTED REPRODUCTION in patients with endometriosis (4). The clinical pregnancy rates were improved when a 2- to 6-month period of hormone suppression with GnRH-a was administered. Given that women with endometriosis may have a lower pregnancy rate in comparison with women with tubal factor infertility (5), some of the reported pregnancy rates in patients pretreated with GnRH-a are rather high (6–8). In a ultralong protocol in which GnRH-a was administered for at least 60 days before ovarian stimulation, a pregnancy rate of 67% was reported (6). In their classic paper, Surrey et al. (7) reported remarkable ongoing pregnancy rates in patients who received a 3-month long-acting GnRH-a regimen when comparing them with those who received conventional midluteal phase GnRH-a down-regulation (80% vs. 53.85%). A similar result was also observed among patients who received treatment with GnRH-a for 6 months before assisted reproduction (8). These facts suggest that prolonged downregulation before IVF treatment might provide superior outcomes in patients with endometriosis. Over two decades ago, it was noted that pregnancy rates might be improved when women are given a period of amenorrhea before embryo transfer (9). High pregnancy rates were found in acyclic or postmenopausal women undergoing oocyte donation and IVF treatment in comparison with the rates among cyclic women. The age of the oocyte donor, the in vitro growth of the embryo, the proportion of defective embryos in culture, and the type of cycle during therapy were found to make no contribution to the difference (10). Edwards suggested that the high pregnancy rates were primarily caused by the previous amenorrhoea in the acyclic or postmenopausal women before the IVF treatment, rather than the use of donor oocytes. Amenorrhea induced by pituitary down-regulation with GnRH-a might also have the same effects. In a study on women undergoing IVF and hormone replacement therapy, cyclic women of their late 40s who were down-regulated for 3 months using GnRH-a were compared with acyclic women and cyclic women of similar age who did not receive GnRH-a pretreatment. A similar pregnancy rate of 30% was found in GnRH-a pretreated women and acyclic women, which was much higher than that of cyclic women without GnRH-a pretreatment (11). The early study suggested that a period of down-regulation might be a favorable factor for infertile women undergoing IVF and that this effect might due to increased endometrial receptivity. If the hypothesis is true, the general IVF population may also benefit from prolonged down-regulation before COH and embryo transfer (ET). However, down-regulation for several months before IVF-ET might not be acceptable in general patient cohort. In this study, we determined the effect of a modified long protocol with 28 days of GnRH-a down-regulation on the live-birth rate in fresh ET cycles in comparison with the long protocol.
MATERIALS AND METHODS Study Participants Institutional review board approval was obtained for this retrospective study. The retrospective analysis was performed on fresh ET cycles stimulated with agonist long protocol in 2
our center between June 2010 and December 2011. All patients had undergone a full set of infertility evaluation and were diagnosed as tubal factor infertility. Presence or absence of endometriosis was confirmed by laparoscopy, and endometritis cases were also excluded from the study. The patients whose male counterpart suffered from male infertility and required intracytoplasmic sperm injection (ICSI) treatment were also excluded. Patients were selected if they met all the following inclusion criteria: women with good physical and mental health, aged <35 years; regular menstrual cycles ranging from 25 to 35 days; body mass index (BMI) <28 kg/ m2; normal basal serum follicle-stimulating hormone (FSH) (%12 IU/L) and estradiol (E2) (%75 pg/mL) levels determined on day 3 of the cycle previous to COH; no uterine abnormalities (fibroids, adenomyosis, m€ ullerian malformations) were assessed by vaginal ultrasound. Patients had not received any ovulation-induction drug within 1 month before COH. The enrollment of patients for each protocol was simultaneous. The treatment assignment was primary decided by the physicians. Before a cycle was initiated, the patient was informed of detailed information of both protocols, including the duration of the down-regulation, the pregnancy rate in previous cycles, and the potential risk of strong pituitary suppression. Based on this information, patients made the decision to accept or reject a specific protocol. All the patients enrolled gave written informed consent for the procedures. The study criteria was met by 955 initiated cycles. Eight initiated cycles were canceled before oocyte retrieval (four in the prolonged protocol, and four in the long protocol). Thirteen cycles in the prolonged protocol group and eight cycles in long protocol group were canceled because no embryo was available. In the prolonged protocol group, 65 cycles and 61 cycles in long protocol were canceled for fresh ET because all embryos were cryopreserved. Because prolonged down-regulation is supposed to affect the endometrial receptivity, and live-birth rates are the main outcome measure in our present study, all cycles canceled for fresh ET were excluded. Therefore, 378 patients receiving the prolonged pituitary down-regulation with GnRH-a before ovarian stimulation and 422 patients receiving the GnRH-a long protocol were included (Fig. 1). The total cancellation rates of ET in the two cohorts were comparable (17.8% in prolonged protocol vs. 14.7% in long protocol; P¼ .197).
Treatment Protocol In the GnRH-a prolonged protocol (prolonged protocol), pituitary down-regulation was obtained with standard full dose (3.75 mg) of triptorelin depot (Decapeptyl 3.75; Ipsen Pharma) in the early follicular phase. The size and number of the antral follicles were monitored from day 1 or 2 of the menstrual cycle. A single intramuscular injection of GnRH-a depot was given when the diameter of the antral follicles was <6 mm. Down-regulation was confirmed after 28 days (no ovarian cysts >8 mm; E2 <50 pg/L) before the gonadotropin stimulation started. No further GnRH analogue treatment was given during the stimulation. It was previously demonstrated elsewhere that LH suppression is maintained until week 8 after the injection of a single depot of triptorelin (12). VOL. - NO. - / - 2014
Fertility and Sterility®
FIGURE 1
mean diameter. We then injected 5,000–10,000 IU of human chorionic gonadotropin (hCG; Lizhu Pharma) intramuscularly. Oocyte retrieval was scheduled for 34 to 36 hours after hCG administration. Oocyte retrieval was performed using a transvaginal ultrasound approach. Semen was produced by masturbation, and motile spermatozoa were prepared by the swim-up procedure. Oocytes were inseminated 4 to 5 hours after collection, pronuclei were identified 17 to 18 hours later, and ET was performed 68 to 70 hours after insemination. Luteal phase support was sustained with natural progesterone in oil (progesterone; XianJu), 60 mg injected intramuscularly each day from the day of oocyte retrieval. A pregnancy test (serum b-hCG determination) was performed 14 days after ET. Clinical pregnancy was defined as the presence of one or more gestational sacs detected on ultrasound scan performed 4 weeks after ET.
Statistical Analysis Analysis was performed with SPSS (version 19; IBM). Independent samples t-test or the Mann-Whitney test was used for continuous variables. Dichotomous variables were analyzed by chi-square test or Fisher's exact test, as appropriate. A multivariable logistic regression model was used to evaluate the contribution of prolonged down-regulation to the difference in live-birth rates. In all analyses, P< .05 was considered statistically significant.
RESULTS Flow sheet describing dropout of enrolled patients. Ren. Prolonged GnRH-a regimen improves outcome. Fertil Steril 2014.
In the GnRH-a long protocol (long protocol), one-third dose (1.25 mg) of triptorelin depot in a single intramuscular injection in the midluteal phase (day 21) of the menstrual cycle preceding treatment was used for pituitary downregulation. Down-regulation was confirmed after 14 days following the same criteria described in the prolonged protocol and was followed by gonadotropin stimulation. In all treatment protocols, patients received two to three ampules (150–225 IU) of gonadotropin per day during the gonadotropin stimulation. A starting dose of 150 IU was given to patients with a high antral follicular count (AFC) (>10) and low body weight (<60 kg). Recombinant FSH (Gonal F; Merck Serono) or domestic urinary human menopausal gonadotropin (hMG; Lizhu Pharma) was used for the gonadotropin stimulation. The serum LH level was determined with the use of a commercial enzyme immunoassay kit before the beginning of ovarian stimulation. When the LH level was <1 IU/L, the patients were stimulated with hMG alone, and otherwise with FSH alone. We adjusted the initial and ongoing dosage according to the patient's age, AFC, BMI, and follicular growth response. During the treatment, ovarian response was monitored by measurements of follicular growth via transvaginal ultrasound and serum E2 levels every 1 to 3 days. Gonadotropin stimulation continued until ultrasonography revealed at least one follicle measuring R18 mm in VOL. - NO. - / - 2014
The average age of the 800 women enrolled in the present study was 30.37 3.49 years (mean standard deviation [SD]), and the mean duration of infertility was 4.06 3.79 years (mean SD). No statistically significant difference was noted in age, infertile period, or BMI in comparing the two groups (Table 1). The basal serum hormone levels were also comparable between the patient cohorts, except for the FSH level. Patients receiving prolonged protocol showed a statistically significantly higher basal FSH level. A statistically significant difference was found in the total doses taken for ovarian stimulation (P¼ .0001); more doses were needed for hyperstimulation in the prolonged protocol group than in the long protocol group, although there was no statistically significant difference in the total days taken for ovarian stimulation (P¼ .235). On the first day of gonadotropin stimulation, the serum LH and E2 level was lower in the prolonged protocol group, which suggested a stronger suppression on pituitary and ovary. On the day of hCG, the serum LH level of prolonged protocol remained lower. However, in terms of the serum level of E2 and P, there were no differences between the two groups. The numbers of AFC on first day of stimulation, oocytes retrieved, fertilized, cleaved, clinical pregnancy, implantation rate, early miscarriage rate, and live-birth rate in both treatment protocols are given. There were greater AFC, oocytes retrieved, and fertilized and cleaved eggs in the long protocol group, but the total numbers of high-quality embryos were similar in both groups. Endometrial thickness on the day of hCG administration was significantly thicker in the prolonged protocol group. There was no difference in the number of 3
ORIGINAL ARTICLE: ASSISTED REPRODUCTION
TABLE 1 Basic characteristics and stimulation variables of the two groups.
Patient characteristics Age (y) Duration of infertility (y) Body mass index Stimulation characteristics Length of stimulation (d) Gonadotropin units (IU) FSH on day 3 (mIU/mL) LH on day 3 (mIU/mL) E2 on day 3 (pg/mL) Antral follicular count on 1st day of stimulation FSH on day 1 of stimulation (mIU/mL) LH on day 1 of stimulation (mIU/mL) E2 on day 1 of stimulation (pg/mL) E2 on day of hCG (pg/mL) LH on day of hCG (mIU/mL) Progesterone on day of hCG (ng/mL) Endometrial thickness on day of hCG (mm) No. of oocytes retrieved No. of oocytes fertilized No. of oocytes cleaved No. of high-quality embryos No. of embryos transferred Cycle outcomes Clinical pregnancy per ET (%) Implantation rate Early abortion rate (%) Live birth per ET (%)
Prolonged (n [ 378)
Long (n [ 422)
P value
29.44 2.82 3.5 [2–5] 20.94 2.60
29.22 3.05 3 [2–5] 21.04 2.58
.315 .763 .446
10.53 1.60 29.61 6.21 6.36 1.94 4.32 2.02 36.11 19.78 8.21 3.44 2.47 1.27 0.85 0.44 12.43 9.35 2,654.76 1,318.26 0.85 0.80 0.92 0.54 1.11 0.23 9.61 4.21 7.45 3.91 5.85 3.23 3 [1–5] 1.95 0.38
10.59 1.82 26.40 6.97 5.45 1.40 4.79 3.02 37.66 27.39 9.95 2.98 1.93 1.21 1.24 0.67 13.29 6.80 2,760.88 1,213.30 1.22 1.00 0.85 0.47 1.06 0.18 10.73 4.01 8.23 3.62 6.66 3.11 3 [2–6] 1.98 0.26
.805 .001 .001 .354 .777 .001 .001 .001 .001 .133 .000 .136 .017 .001 .002 .001 .074 .264
242/378 (64.02) 50.27% 22/242 (9.09) 210/378 (55.56)
240/422 (56.87) 39.69% 31/240 (12.92) 193/422 (45.73)
.039 .001 .179 .006
Note: Numbers are mean þ standard deviation (range) or median [interquartile range]. E2 ¼ estradiol; ET ¼ embryo transfer; FSH ¼ follicle-stimulating hormone; hCG ¼ human chorionic gonadotropin; LH ¼ luteinizing hormone. Ren. Prolonged GnRH-a regimen improves outcome. Fertil Steril 2014.
embryos transferred. There were higher rates of clinical pregnancy (64.02% vs. 56.87%, P¼ .039) and implantation (50.27% vs. 39.69%, P< .001) in the prolonged protocol group. There was no statistically significant difference between the groups in the spontaneous abortion rate. The live-birth rate in the prolonged protocol group was statistically significantly increased (55.56% vs. 45.73%, P¼ .006). To evaluate the contribution of the period of downregulation to the difference in live-birth rate, a multivariate logistic regression model was used. The variables that were different between the two patient cohorts (basal FSH levels, AFC, FSH, LH, and E2 levels on the day of stimulation, LH levels and endometrial thickness on the day of hCG, number of oocytes retrieved) were selected as potential confounding factors. Other factors (age, progesterone elevation on the hCG day, number of good quality embryo) were also selected based on clinical experiences, although they were similar in both cohorts. The Hosmer-Lemshow goodness-of-fit chi-square test statistic was 8.75 (P¼ .364), which suggested that the multivariable model was a good fit (P>.05). Adjusted by the previously mentioned potential confounding factors, the odds ratio of prolonged down-regulation versus 14-day down-regulation on the live-birth rate was 1.464 (95% confidence interval, 1.069–2.131).
DISCUSSION Several studies using prolonged GnRH-a pretreatment before IVF have obtained relatively high pregnancy rates (6–8). 4
However, in these studies the sample sizes of each group were small, and the pathology of endometriosis was a major confounding factor. In a randomized controlled trial involving 60 patients, Fabregues et al. (13) reported that monthly prolonged down-regulation with GnRH-a did not improve outcomes in women who did not have endometriosis. Their sample size calculation was based on the assumption that a 30% difference in favor of long-term down-regulation versus controls existed, which was obtained from a study with women aged in their late 40s. For normogonadotropic women of reproductive age, it is unlikely that a 30% improvement in pregnancy rate can be achieved solely by long-term downregulation. To detect or reject the existence of a smaller difference might require a larger sample size. Our data argue that prolonged down-regulation still has a beneficial effect on the live-birth rate. The effect of prolonged down-regulation also has been observed in women who do not achieve timely suppression with daily dosed GnRH-a. Loutradis et al. (14) compared the IVF outcomes of prolonged GnRH-a in a small group of patients whose administration period was shorter or longer than 15 days, and found a favorable effect of GnRH-a prolongation on embryo cleavage speed and pregnancy rate. Later, these investigators reported that no difference in cycle outcome or clinical pregnancy was found between women receiving a 14- to 24-days' or 25- to 40-days' desensitization (15). More recently, Dessolle et al. (16) reported similar livebirth rates per transfer (28.86% vs. 32%) in women receiving VOL. - NO. - / - 2014
Fertility and Sterility® 14-days of down-regulation and in women who needed 7 to 28 additional days to achieve complete suppression. These studies suggested that prolonging GnRH-a might not compromise the outcomes even if it did not provide a favorable effect. However, it is worth bearing in mind that these reports were based on patient cohorts who had failed to achieve satisfactory suppression in a standard-duration down-regulation protocol; why they required a longer GnRH-a course is not fully understood. Our data showed that in the cohort with prolonged downregulation, the endometrial thickness was statistically significantly higher. The thickness of the endometrium has been used as a marker of adequate receptivity of the uterus and as a prognostic factor in ET. Richter et al. (17) demonstrated a significant increase in the pregnancy rate as the endometrial thickness increased, independent of the number or quality of the embryos transferred. Their conclusion was confirmed by Al-Ghamdi et al. (18) in a 2,464-cycle cohort study. More recently, Chen et al. (19) reported results that were closely similar to those two studies. It is well known that endometrial growth and differentiation are sensitive to steroid hormones; Edwards (9) hypothesized that steroid-responsive systems in the uterus become impaired after years of regular menstrual cycles, so a period of amenorrhea or down-regulation might recover the systems from this constant stimulus. A greater thickness of endometrium achieved by prolonged downregulation might support the hypothesis of endometrial recovery. Prolongation of down-regulation also results in enhanced exposure to GnRH-a. The GnRH-a may contribute to the outcomes by an effect on the embryo or a direct effect on the endometrium. The assessment of embryo quality is difficult to establish, but after long suppression of the pituitary-ovarian axis, the function of oocytes or intrafollicular dynamics might be induced and improve embryo quality. A study in an animal model suggested that preimplantation embryonic development was significantly enhanced by incubation with increasing concentrations of GnRH-a, and noted that the expression of GnRH and its receptor were also observed in human preimplantation embryos (20). Previous studies suggested that GnRH-a treatment may have a direct effect on endometrium by regulating the expression of the enzymes and cytokines (21–23). Intracellular signals of the transforming growth factor-b pathway, interleukin-1b and vascular endothelial growth factor production, and extracellular-signal-related kinase activation in human endometrial cells were all found to be affected by GnRH-a binding (24, 25). In mice, the expression of endometrial receptivity markers such as integrin-b3 subunit and leukemia inhibitory factor (LIF) is increased in ovarian stimulation with GnRH-a (26). However, direct evidence supporting the role of GnRH-a in human embryo implantation remains limited. Schachter et al. (27) reported in a randomized study that administering GnRH-a before oocyte retrieval in GnRH antagonist cycles may improve pregnancy rates. In an oocyte donation program, after a single dose of 0.1 mg triptorelin administered 3 days after ET (28), a higher implantation rate was obtained compared with the control group who received VOL. - NO. - / - 2014
placebo at the same time. The same investigators later reported similar results in both GnRH-a- and antagonisttreated ovarian stimulation cycles (29). Studies with frozen embryo transfer (FET) cycles provide an opportunity to estimate the effects of GnRH-a on oocyte and endometrium separately. It suggests that the embryos derived from GnRH-a cycles might not be superior to those from GnRH antagonist cycles, as the outcomes in subsequent FET cycles are comparable (30–33). In contrast, administering GnRH-a before ET in artificial FET cycles might improve pregnancy rates in comparison with natural cycles (34) or artificial cycles without GnRH-a (35). Although the role of GnRH-a in endometrium preparation remains controversial (36), the collective findings in fresh and FET cycles suggest a role for GnRH-a in improving embryo implantation. In our study, a full-dose depot GnRH-a injection was used to achieve suppression in the prolonged protocol. Although the formulation was primarily used to provide pituitary suppression for 28 days, a previous study demonstrated that a full-dose depot GnRH-a injection was sufficient to maintain LH suppression until week 8 after the injection, and estrogen secretion started to be restored in the course of weeks 7–8. Suppression of pituitary and ovarian function appears to be continued until week 8 after the injection (12). In line with previous findings, our data showed that an LH surge was prevented until the day of oocyte retrieval and that no additional GnRH analogues were needed during ovarian stimulation. However, full-depot GnRH in the long protocol was also associated with strong pituitary suppression (37), which might impair the ovarian response to exogenous FSH. Studies have shown that a half-dose (37) or one-third dose (38) depot triptorelin injection was sufficient to prevent LH surges and to improve the ovarian response. In comparison with those down-regulated with one-third dose depot triptorelin for the long protocol, the prolonged protocol required more exogenous gonadotropin and yielded fewer oocytes. However, the number of good quality embryos was unaffected, and the live-birth rate was significantly higher. The benefit of prolonged down-regulation might have partially balanced the effect of the drawback of strong suppression. However, in our present study, the participants were limited to young, normogonadotropic women. For patients who have responded poorly in previous cycles or patients with predictors of poor response, loss of ovarian response after prolonged downregulation could become a significant issue. In which case, alternative protocols should be taken into consideration. A reduced LH level after strong pituitary suppression may be detrimental to oocyte quality and uterine receptivity (39). When LH levels were <0.5 IU/L, this led to significantly lower E2 production, fewer oocytes, a lower fertilization rate, and fewer embryos available for cryopreservation (40–42). An increased risk of early pregnancy loss has also been suggested (42). The work of Dal Prato et al. (37) has suggested that low LH levels associated with full-dose depot GnRH-a in a long protocol may affect the clinical outcomes in patients stimulated with recombinant FSH. In contrast, a similar randomized trial in which patients were stimulated with hMG failed to find the same association (43). The difference between the results of the two studies probably lies in the 5
ORIGINAL ARTICLE: ASSISTED REPRODUCTION gonadotropin used. Because hMG contains nearly equal activity of FSH and LH, it might contribute as a source of exogenous LH supplementation. The addition of exogenous LH in patients whose serum LH has been suppressed might increase the number of developmentally competent oocytes as well as the rate of implantation (39). In our present study, patients with a serum LH of <1 IU/l might also have benefited from hMG stimulation. The nonrandomized design is a significant weakness in our study. Although a nonrandomized trial has its own advantages, it is always associated with selection bias issues. With selection bias being introduced, one might expect the result will be skewed in some ways. For example, patients with good ovarian response might be more likely to be assigned to the prolonged group, in which the suppression is stronger. However, a higher basal FSH level in prolonged protocol might suggest the opposite. Therefore, adjusting for covariates is important for interpreting the results. Methodologic study suggests that adjusted results from nonrandomized studies with a rich set of covariates can approximate results from randomized experiments (44). Although the unknown and unmeasured confounders remained to be unadjusted for in our study, the results establish a crude baseline that can be compared with the results of subsequent studies. In conclusion, our data suggest that a prolonged ovarian suppression of 28 days with a standard full dose before ovarian stimulation in IVF-ET might improve the live-birth rate per fresh ET. The effects might be associated with increased endometrial receptivity during embryo implantation, although the precise mechanism remains unclear. The study is limited by its retrospective nature as well as the specified patient cohort. The patient cohort consisted of young, normogonadotropic women. Based on previous reports (9), a more profound effect on endometrium might be expected in patients of advanced age. However, a decrease in ovarian reserve and oocyte quality are also age related. Whether prolonged down-regulation for 28 days can improve the livebirth rate among women of advanced age is unknown. Acknowledgments: The authors thank all the staff, nurses, and physicians at the Reproductive Medicine Center for their support in generating this manuscript.
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