ORIGINAL ARTICLE: ASSISTED REPRODUCTION

Dual trigger with gonadotropin-releasing hormone agonist (GnRHa) and standard dose human chorionic gonadotropin (hCG) to improve oocyte maturity rates Daniel Griffin, M.D.,a Richard Feinn, Ph.D.,b Lawrence Engmann, M.D.,a John Nulsen, M.D.,a Tara Budinetz, D.O.,a and Claudio Benadiva, M.D., H.C.L.D.a a

Center for Advanced Reproductive Services, Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, and b Department of Biostatistics, University of Connecticut Health Center, Farmington, Connecticut

Objective: To evaluate the percentage (%) of mature oocytes retrieved in patients with a previous history of >25% immature oocytes retrieved who were triggered with gonadotropin-releasing hormone agonist (GnRH-a) and human chorionic gonadotropin (hCG) to induce oocyte maturation. Design: Retrospective cohort study. Setting: A university-based tertiary fertility center. Patient(s): Patients with a history of >25% immature oocytes retrieved in a prior in vitro fertilization cycle who were triggered with GnRH-a and hCG 5,000 IU or 10,000 IU in a subsequent cycle from January 2008 through February 2012. Intervention(s): Dual trigger of GnRH-a and hCG 5,000 or 10,000 IU. Main Outcome Measure(s): Percent of mature oocytes retrieved and fertilization rate. Result(s): The proportion of mature oocytes retrieved was significantly higher with a dual trigger compared with the subject's previous cycle (75.0%, interquartile range 55.6%–80.0% vs. 38.5%, interquartile range 16.7%–55.6%). The odds of a mature oocyte retrieved for patients who received a dual trigger was 2.51 times higher after controlling for stimulation protocol, hCG dose, gonadotropin dose, and oocyte retrieval time interval (odds ratio 2.51; confidence interval 1.06–5.96). The implantation, clinical, and ongoing pregnancy rates for the dual trigger were 11.8%, 26.1%, and 17.4%, respectively. Conclusion(s): In patients with a low percentage of mature oocytes retrieved who are triggered with a combination of GnRH-a and hCG, the % of mature oocytes retrieved improved. in vitro fertilization outcomes, however, remain poor, suggesting an underlying oocyte dysfunction. (Fertil SterilÒ 2014;-:-–-. Use your smartphone Ó2014 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Dual trigger, gonadotropin-releasing hormone agonist, oocyte maturity, in vitro and connect to the fertilization Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/griffind-dual-trigger-gnrha-hcg-oocyte-maturity-rates/

I

n vitro fertilization involves stimulating the ovaries with gonadotropins to yield mature oocytes that are

capable of fertilization. Oocyte maturation occurs in vivo after the luteinizing hormone (LH) surge during the menstrual

Received February 19, 2014; revised April 4, 2014; accepted April 16, 2014. D.G. has nothing to disclose. R.F. has nothing to disclose. L.E. has nothing to disclose. J.N. has nothing to disclose. T.B. has nothing to disclose. C.B. has nothing to disclose. Reprint requests: Claudio Benadiva, M.D., H.C.L.D., The Center for Advanced Reproductive Services, Dowling South Building, 263 Farmington Avenue, University of Connecticut Health Center, Farmington, Connecticut 06030-6224 (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.2014.04.028 VOL. - NO. - / - 2014

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cycle. The oocyte completes meiosis I and stops at metaphase II until fertilization, when meiosis II is completed (1). Oocyte maturation for in vitro fertilization cycles is commonly induced by human chorionic gonadotropin (hCG) as a surrogate for the natural LH surge. After controlled ovarian stimulation, some of the oocytes retrieved are arrested at the germinal vesicle or metaphase I stage despite correct administration of hCG. 1

ORIGINAL ARTICLE: ASSISTED REPRODUCTION Patients who have >25% of immature oocytes retrieved have poorer IVF outcomes compared with patients who have a larger proportion of mature oocytes obtained (2, 3). Low oocyte maturity or the inability to retrieve mature oocytes has severe consequences, including having very few or no embryos available for transfer, which reduces the chance of conceiving with IVF. Following the introduction of GnRH antagonists in clinical practice, the use of a gonadotropin-releasing hormone agonist (GnRH-a) bolus to induce an endogenous LH surge has been explored in many clinical scenarios, primarily for prevention of ovarian hyperstimulation syndrome (OHSS; 4). Among the advantages of using a GnRH-a to trigger oocyte maturation is the induction of a more physiologic release of LH and follicle-stimulating hormone (FSH), resembling what occurs in the natural menstrual cycle (4). In addition, some studies have suggested an increase in the percentage of mature oocytes retrieved when triggered with GnRH-a compared with hCG (4, 5). Shapiro et al. (6) introduced the concept of ‘‘dual trigger’’ with a combination of GnRH-a and a low-dose hCG, primarily for the purpose of OHSS prevention. This approach was subsequently corroborated by Griffin et al. (7). More recently, Castillo et al. (8) reported the successful use of a dual trigger with GnRH-a and hCG in a patient showing repetitive immature oocytes and empty follicle syndrome. We hypothesized that the endogenous LH and FSH released by the GnRH-a bolus, in addition to the hCG, would result in a larger proportion of mature oocytes in patients with a history of poor oocyte maturation after the hCG trigger alone. As far as we are aware, this is the first study showing a series of patients in whom a dual trigger with GnRH-a plus a standard dose of hCG has been applied as a potential treatment in patients with a history of a large percentage of immature oocytes retrieved, and compared with an hCG trigger alone in a previous cycle.

MATERIALS AND METHODS A retrospective chart review was performed on all patients who received the dual trigger of a GnRH-a and a standard dose of hCG for oocyte maturation during their IVF cycle from January 2008 until February 2012. Institutional review board approval was obtained at the University of Connecticut Health Center. Patients who were eligible for the study had a prior IVF cycle with >25% immature (germinal vesicle or metaphase I) oocytes retrieved, and a subsequent IVF cycle using a dual trigger to induce oocyte maturation. All patients must have used the intracytoplasmic sperm injection (ICSI) technique for fertilization, so that the oocyte maturation could be assessed. Exclusion criteria included patients who used a standard IVF insemination technique for oocyte fertilization. Patients underwent various IVF stimulation protocols including microdose leuprolide (9) or pituitary down regulation with leuprolide as previously described (10) during their prior cycle, and this was compared with the dual-trigger cycle. Stimulation protocols were selected based on physician's preference. During the previous cycle, patients were triggered with hCG only. The dose of hCG was either 5,000 IU or 10,000 IU. 2

For the dual-trigger cycle, all patients used a GnRH antagonist controlled ovarian stimulation protocol. All patients presented for a baseline transvaginal ultrasound on day 2 of their menstrual cycle and started recombinant FSH (rFSH; Follistim; Organon USA Inc. or Gonal-F; Serono Inc) with or without human menopausal gonadotropin (Menopur; Ferring Pharmaceuticals). The starting gonadotropin dose of 225–600 IU was based on patient age, body mass index, day-3 FSH, antral follicle count, anti-M€ ullerian hormone level, and prior response to gonadotropins. Patient response was monitored during the IVF cycle with serial transvaginal ultrasounds for follicular measurements and serum E2 levels. The dose of gonadotropins was adjusted according to a patient's response. A daily subcutaneous (SC) dose of 0.25 mg GnRH antagonist (Ganirelix; Organon USA Inc.) was started after a follicle reached R14 mm in diameter or serum E2 level was >350 pg/mL; the dose was continued until the day of the oocyte maturation trigger. Patients were triggered when at least 3 follicles reached R17 mm in diameter, with a combination of 1 mg SC leuprolide acetate plus 5,000–10,000 IU SC hCG. Transvaginal ultrasound-guided oocyte retrieval was performed 35–37 hours after trigger injection. Serum LH, E2, P, and hCG levels were assessed the day after trigger to ensure adequate LH surge response and hCG absorption. Luteal support included 50 mg intramuscular P daily, commencing the day after oocyte retrieval until either a negative pregnancy test or 8 weeks' gestation. Patients had either a cleavage stage or a blastocyst stage embryo transfer (ET) 3 or 5 days after the oocyte retrieval. The type and number of embryos transferred were based on embryo quality and the age of the patient. The primary outcome measures were the number and percentage of mature oocytes retrieved with the dual-trigger protocol, compared with the patient's prior cycle. Secondary outcome measures included fertilization rate, implantation rate, clinical and ongoing pregnancy rates, as well as live birth rate. Implantation rate was defined as the number of gestational sacs, as assessed by ultrasound at 7 weeks' gestation, divided by the number of embryos transferred for each patient. Clinical pregnancy was defined as evidence of fetal cardiac activity on ultrasound at 7 weeks' gestation.

Statistical Analysis Statistical analyses were performed using SPSS Release 21.0 (SPSS Inc.). A Chi-square or Fisher's exact test was used for categorical variables, as appropriate. Wilcoxon's rank sum test was used for continuous variables, as appropriate, between the subject's prior IVF cycle and dual-trigger cycle. A general estimation equation with logistic regression link was used to identify and control for any confounding variables and their effect on the primary outcome. Nonparametric data are presented as the median with interquartile range (25th–75th percentile). All P values are two-sided, and values < .05 indicate statistical significance.

RESULTS The baseline characteristics for the patients are shown in Table 1. The stimulation protocols differed between the two groups. In the dual-trigger cycle, all patients used an VOL. - NO. - / - 2014

Fertility and Sterility®

TABLE 1 Patient demographic characteristics from the dual trigger cycle. Characteristic

Dual trigger (n [ 27)

Age (y) BMI (kg/m2) Baseline serum FSH (IU/L) Baseline serum LH (IU/L) €llerian hormone (ng/mL) Antimu Prior IVF cycles Etiology of infertility Male Unexplained Diminished ovarian reserve Anovulation Endometriosis Tubal Other

37.5  4.3 26.0  5.5 7.4  4.1 3.8  1.4 1.2  1.0 1.7  0.9 9/27 (33.3) 6/27 (22.2) 4/27 (14.8) 2/27 (7.4) 2/27 (7.4) 2/27 (7.4) 2/27 (7.4)

Note: Data presented as mean  SD or n (%), unless otherwise stated. BMI ¼ body mass index; FSH ¼ follicle-stimulating hormone; LH ¼ luteinizing hormone; IVF ¼ in vitro fertilization. Griffin. GnRH-a/hCG trigger for low egg maturity. Fertil Steril 2014.

antagonist protocol. During the prior cycle, 7 of 27 (25.9%) used a microdose leuprolide protocol for stimulation; 8 of 27 (29.6%) used a down regulation leuprolide protocol; and 11 of 27 (40.5%) used an antagonist protocol. This difference was statistically significant between the two groups (P< .01). The average amount of time between the prior cycle and the dual-trigger cycle was 98.8 days (range 28–241 days). The cycle characteristics between the prior IVF cycle and the dual-trigger cycle are shown in Table 2. The total dose of gonadotropins used was significantly higher (P¼ .02) in the dual-trigger cycle (5400 IU [interquartile range 3300–6750 IU]) compared with the previous cycle (4200 IU [interquartile range 2575–6000IU]). The total number of oocytes retrieved (11 [interquartile range 5–16] vs. 9 [interquartile range 4– 14], P¼ .02); number of mature oocytes retrieved (7 [interquartile range 4–9] vs. 3 [interquartile range 1–5], P< .01); and percentage of mature oocytes (75.0% [interquartile range 55.6%–80.0%] vs. 38.5% [interquartile range 16.7%–55.6%], P< .01) were significantly higher in the dual-trigger cycle compared with the prior hCG-triggered IVF cycle. The serum E2 level the day after trigger was significantly lower in the dual-trigger group (1,904 pg/mL [interquartile range 1,176–2,592 pg/mL]) compared with the prior IVF cycle (2,032 pg/mL [interquartile range 1,140–3,827 pg/mL] P¼ .01). The serum P level the day after trigger was significantly higher in the dual-trigger group compared with the prior IVF cycle (4.0 ng/mL [interquartile range 2.9–5.2 ng/mL] vs. 2.6 ng/ mL [interquartile range 1.9–4.0 ng/mL], P< .01). The serum hCG levels the day after trigger were significantly higher in the dual-trigger group compared with the prior IVF cycle (203 IU/L [interquartile range 128–297 IU/L] vs. 135 IU/L [interquartile range 96–190 IU/L], P< .01). At the time of trigger in the dual-trigger group, 25 of 27 patients received 10,000 IU hCG, and 2 of 27 received 5,000 IU hCG. In the prior cycle group, 9 of 27 patients received 5,000 IU hCG, and 18 of 27 received 10,000 IU hCG at the time of trigger. In the dual-trigger group, the time interval between the hCG and the GnRH-a dose and retrieval was either 35 VOL. - NO. - / - 2014

hours (40.7%); 36 hours (29.6%); or 37 hours (29.6%). In the previous cycle, one patient had a 37-hour interval (3.7%), and the remaining patients had a 35-hour interval (96.3%). The time interval was significantly different between the two groups (P< .01). The general estimating equation with logistic regression link determined that the odds of having a mature oocyte retrieved for cycles using a dual trigger was 2.51 times higher than for cycles that did not, after controlling for stimulation protocol, hCG dose, total dose of gonadotropins, and oocyte retrieval time interval (odds ratio 2.51; confidence interval 1.06–5.96). The logistic regression analysis is presented in Table 3. There were 10 of 27 (37.0%) cycles with a positive hCG after an ET. Four of the 10 (40%) patients had a biochemical miscarriage. One of the 10 (10%) had a clinical miscarriage. Four patients did not have an ET after ovum pick-up. The first patient had 12 eggs retrieved, of which 3 were mature with no fertilization after ICSI. The second patient had 2 eggs retrieved, neither of which was mature. Two other patients had all of their embryos frozen electively for preimplantation genetic diagnosis. The implantation, clinical, and live birth rates per ET for the dual trigger were 11.8% (7 of 59); 26.1% (6 of 23); and 17.4% (4 of 23), respectively.

DISCUSSION This study shows that a statistically significant increase in the percentage of mature oocytes retrieved can be achieved when patients received a dual trigger with a GnRH-a plus a standard dose of 5,000–10,000 IU hCG compared with hCG alone. The increase in the percentage of mature oocytes was significant after controlling for the change in protocol, total gonadotropin dose, time interval between trigger injection and oocyte retrieval, as well as the dose of hCG used. A dual trigger in patients with a history of low oocyte maturation is a viable treatment to improve the rate of oocyte maturation. However, despite the improvement in mature oocytes retrieved, the implantation and pregnancy rates remained low, suggesting that these patients may have an underlying oocyte dysfunction. During IVF cycles, patients who have R25% of immature oocytes retrieved have a reduced pregnancy rate (2, 3). The incidence of patients with a high proportion of immature oocytes is unknown. The etiology of oocyte maturation arrest is complex, and to date, no effective treatment options have been described. During the menstrual cycle, ovulation is preceded by a surge of LH and FSH (11). The surge of these two hormones triggers the oocyte maturation process. Oocytes are arrested in meiosis I until the LH/FSH surge, after which the oocyte completes meiosis I and arrests at metaphase II until fertilization takes place (2). Meiosis resumes approximately 18 hours after the LH surge (1), and the surge must be maintained for 14–27 hours for maximum oocyte maturation to occur (12). During an IVF cycle, hCG is given to mimic the LH surge, and oocyte retrieval is generally performed 35–37 hours later. However, hCG has no FSH receptor activity, unlike the GnRHa, which releases an endogenous FSH (and LH) surge. Follicle-stimulating hormone plays a role in oocyte maturation during the natural menstrual cycles and may have a 3

ORIGINAL ARTICLE: ASSISTED REPRODUCTION

TABLE 2 Ovarian stimulation outcomes and endocrine profiles between the dual trigger and prior IVF cycle. Variable Outcome of ovarian stimulation Total days of stimulation Total dose of gonadotropins (IU) Oocytes retrieved (n) Mature oocytes (n) % of mature oocytes Fertilization rate (%) Embryos transferred (n) Endocrine profile Peak E2 (pg/mL) E2, day after trigger (pg/mL) P, day after trigger (ng/mL) LH, day after trigger (IU/L) hCG, day after trigger (IU/L)

Prior cycle (n [ 27)

Dual trigger (n [ 27)

P value

10 (8–11) 4,200 (2,575–6,000) 9 (4–14) 3 (1–5) 38.5 (16.7–55.6) 66.7 (40.0–100.0) 1 (0–2)

10 (9–11) 5,400 (3,300–6,750) 11 (5–16) 7 (4–9) 75.0 (55.6–80.0) 83.3 (72.4–93.8) 2 (2–3)

NS .02 .02 < .01 < .01 NS NS

1,490 (1,142–2,752) 2,032 (1,140–3,827) 2.6 (1.9–4.0) NA 135 (96–190)

1,661 (1,163–2,162) 1,904 (1,176–2,592) 4.0 (2.9–5.2) 54.2 (36.0–81.4) 203 (128–297)

NS .01 < .01 NA < .01

Note: NS ¼ not significant; LH ¼ luteinizing hormone; hCG ¼ human chorionic gonadotropin; NA ¼ not applicable. Griffin. GnRH-a/hCG trigger for low egg maturity. Fertil Steril 2014.

benefit in patients with infertility treated with IVF. FSH has been shown to be important in in vitro maturation of oocytes (13) and to induce ovulation independent of the LH surge in animal studies (14). The FSH surge induces LH receptor formation on luteinized granulosa cells, and promotes oocyte maturation and cumulus expansion (4). A randomized controlled trial has shown that a bolus of FSH given at the time of hCG to trigger oocyte maturation improved fertilization rates in IVF, but did not show a statistically significant change in pregnancy rates (15). Treatment options for patients with a high proportion of immature oocytes include in vitro maturation, changing the timing of oocyte retrieval after hCG trigger, or triggering with a GnRH-a to improve in vivo maturation of oocytes. In vitro maturation of human oocytes could be considered a potential treatment for patients with a large percentage of immature oocytes retrieved (16); it has been used to reduce the risk of OHSS in women with PCOS and for fertility preservation (17). This technology is still evolving and is considered experimental (17). Timing of oocyte retrieval after hCG injection has been studied to maximize the number of mature oocytes retrieved before release of the oocyte from the follicle (18). The best timing for oocyte retrieval remains controversial. Nargund et al. (19) showed no difference in IVF outcomes between oocyte

retrieval at 33–41 hours after hCG injection. Other studies have shown improved oocyte maturation with a 38-hour interval (18, 20). However, a meta-analysis showed no difference in fertilization rate, implantation rate, or pregnancy rate after increasing the interval from hCG trigger to oocyte retrieval (18). Timing of oocyte retrieval is another tool to improve oocyte maturation, but it may not improve pregnancy outcomes. A GnRH-a trigger has been used to induce oocyte maturation for IVF (4). In addition to the endogenous surge of LH as well as FSH which leads to oocyte maturation (4, 12, 14) GnRH has receptors on the ovarian granulosa cells, which have been shown to play a role in regulating ovulation (21). A combination of the actions from LH, FSH, and GnRH may have a beneficial effect in improving oocyte maturation in patients with a history of low oocyte maturity. Given alone for triggering oocyte maturation, GnRH-a causes corpus luteum dysfunction, decreased pregnancy rates, and increased miscarriage rates (5, 22). The simultaneous administration of hCG negates the luteolytic effects of the GnRH-a trigger, providing adequate luteal support and improving pregnancy rates (23). The GnRH-a trigger has been used successfully for treatment of empty follicle syndrome (24, 25), and a recent case report described a successful pregnancy with a dual GnRH-a and hCG trigger in a patient with a history of empty follicle syndrome and

TABLE 3 General estimation equation with logistic regression link analysis. Parameter Stimulation protocol Antagonist Down regulation Microdose leuprolide hCG to VOR time interval, h hCG dose, IU Total dose of gonadotropins, IU Dual trigger

B 0.52 0.51 Reference group 0.46 0.22

Dual trigger with gonadotropin-releasing hormone agonist and standard dose human chorionic gonadotropin to improve oocyte maturity rates.

To evaluate the percentage (%) of mature oocytes retrieved in patients with a previous history of >25% immature oocytes retrieved who were triggered w...
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