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The different impact of stimulation duration on oocyte maturation and pregnancy outcome in fresh cycles with GnRH antagonist protocol in poor responders and normal responders
Taiwanese Journal of Obstetrics & Gynecology 58 (2019) 471e476
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Original Article
The different impact of stimulation duration on oocyte maturation and pregnancy outcome in fresh cycles with GnRH antagonist protocol in poor responders and normal responders Yu-Chieh Yang a, Yi-Ping Li b, Song-Po Pan a, Kuang-Han Chao a, Chin-Hao Chang c, Jehn-Hsiahn Yang a, Shee-Uan Chen a, * a b c
Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, Taiwan Department of Obstetrics & Gynecology, Shin Kong Memorial Wu Ho-Su Hospital, Taipei, Taiwan Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 14 March 2019
Objective: To study the impact of stimulation duration on intracytoplasmic sperm injection (ICSI) embryo transfer (ET) outcome in poor and normal responders during controlled ovarian stimulation using gonadotropin-releasing hormone (GnRH) antagonist protocol. Materials and methods: This is a retrospective cohort study. There were 1481 women undergoing ICSI-ET cycles. Women with ovum pick-up number 3 were defined as poor responders (n ¼ 235), and those with a number 4 were normal responders (n ¼ 1246). Results: The mean stimulation duration was shorter in poor responders with pregnancy group as compared with normal responders with pregnancy group (7.8 ± 2.2 vs. 9.2 ± 1.6 days, p < 0.01). Poor responders with a shortest stimulation duration (6 days) appeared a higher live birth rate (6 days: 33.3%, 7e8 days: 20.0%, 9e10 days: 15.9%, and 11 days: 11.1%, p ¼ 0.18). Normal responders with a shortest stimulation duration (6 days) appeared a lowest live birth rate (6 days: 28.6%, 7e8 days: 35.8%, 9e10 days: 33.6%, and 11 days: 29.3%, p ¼ 0.61). Oocyte maturation rate was significantly lower at stimulation durations 6 days group (6 days: 67%, 7e8 days: 80%, 9e10 days: 85%, and 11 days: 87%, p ¼ 0.02) in normal responders. Conclusion: In ICSI-ET cycles, stimulation duration appears to have different impact on oocyte maturation, clinical pregnancy rates and live birth rates in both poor and normal responders. © 2019 Taiwan Association of Obstetrics & Gynecology. Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: GnRH antagonist Controlled ovarian stimulation Poor responder Normal responder Stimulation duration
Introduction The long protocol of gonadotropin-releasing hormone (GnRH) agonist in combination with gonadotropin has been traditionally used for controlled ovarian stimulation (COS) in in-vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSIET) treatments [1]. In recent years, the GnRH antagonist protocol becomes increasingly utilized due to its advantage in being patientfriendly and in reduction of ovarian hyperstimulation syndrome (OHSS) [2e7]. An updated meta-analysis comparing the GnRH antagonist protocol and conventional GnRH agonist long protocol
* Corresponding author. Department of Obstetrics and Gynecology, National Taiwan University Hospital, No. 8 Chung-Shan South Road, Taipei, Taiwan. Fax: þ886 2 23116056. E-mail address: [email protected] (S.-U. Chen).
had shown a comparable pregnancy rate and reduction in OHSS [2]. The GnRH antagonist protocol has been reported to have shorter stimulation duration than GnRH agonist protocol [8,9]. However, the effect of gonadotropin stimulation duration on IVF/ICSI outcome in the GnRH antagonist protocol has not been clearly studied. The duration of gonadotropin stimulation is associated with follicular growth, oocyte maturation, oocyte quality and endometrial development. Insufficient period of gonadotropin exposure may lead to nuclear or cytoplasmic immaturity of oocytes [10,11]. On the other hand, prolonged stimulation duration may cause postmaturity of oocytes or apoptosis of granulosa cells and oocytes [12]. It may also result in elevated progesterone (P4) and estradiol (E2) levels and impaired endometrial receptivity [13e15]. Various criteria, including follicular size and estradiol levels, have been
https://doi.org/10.1016/j.tjog.2019.05.007 1028-4559/© 2019 Taiwan Association of Obstetrics & Gynecology. Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
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applied in determining the time of triggering final oocyte maturation [13,16,17]. Only few studies have investigated the correlation between the length of follicular phase and IVF outcome [11,18,19]. The cycles analysed in these reports were mostly GnRH agonist long protocol, and their results were inconclusive. Martin et al. reported no statistically significant difference in clinical pregnancy rates among women with different stimulation durations [18]. Mardesic et al. reported that women with short duration of stimulation of only 6e8 days have the same chance of pregnancy as those with a longer duration of stimulation [20]. Another study by Chuang et al. showed a worse pregnancy outcome with a longer stimulation duration [19]. Due to their different mechanisms and longer gonadotropin stimulation in the long GnRH agonist protocol, these studies might not be applicable to GnRH antagonist cycles. Besides, poor ovarian responders and normal responders may even have different patterns of follicular recruitment and growth rate [10,21e23]. Therefore, they might need to be analyzed separately. In this study, we aim to investigate the impact of stimulation duration of gonadotropin on oocyte maturity, hormonal milieu relating to endometrium, pregnancy rates and live birth rates in the GnRH antagonist cycles. The differences in the effect of stimulation duration on IVF outcomes between poor ovarian responders and normal responders are explored. This study could be helpful in understanding the influence of duration of ovarian stimulation on the IVF outcomes using the GnRH antagonist protocol. Materials and methods Study population This was a retrospective, single-center cohort study, and was approved by the Research Ethics Committee of National Taiwan University Hospital (NTUH). Patients were treated with routine practice of assisted reproductive technologies using the flexible GnRH antagonist protocol [10,24] from January 2009 to June 2017 at NTUH reproductive medicine center. The data were collected from medical records and the computerized database. The inclusion criteria were: eligible indications for COS before ICSI due to male factor using a GnRH-antagonist protocol. Only the first stimulated ICSI cycles without pretreatment were included for analysis in this study. The exclusion criteria were: women with uterine-factor infertility; canceled cycles that did not reach oocyte retrieval; oocyte frozen or oocyte donation cycles; and embryos all frozen due to risk of OHSS. Patient characteristics, including age, body mass index (BMI), and basal hormone levels were recorded. Clinical parameters, including stimulation duration of gonadotropin, gonadotropin dosage, hormone levels at human chorionic gonadotropin (hCG) day, fertilization method, and number of embryos transferred were documented. Other parameters, including number of oocytes retrieved, maturation rate of oocytes, fertilization rate, good embryo rate, implantation rate, biochemical pregnancy rate, clinical pregnancy rate, and live birth rate were analyzed. Study end-points were oocyte maturation rate, clinical pregnancy rate, and live birth rate. Oocyte maturation rate, expressed as metaphase II (MII) oocytes per retrieved oocytes were calculated and analyzed. The patients were categorized according to their ovarian response as poor responders and normal responders. In this study, poor responders were defined as ovum pick-up number 3. To further demonstrate the effect of stimulation duration on each clinical variable, both poor and normal responders were categorized into four groups according to the stimulation durations (6 days, 7e8 days, 9e10 days, and 11 days) to reflect a clinically applicable length of stimulation duration.
Protocols for COS A flexible GnRH antagonist protocol (GnRH antagonist: Orgalutran®, ganirelix; Organon, Dublin, Ireland, or Cetrotide®, cetrorelix acetate; Merck-Serono, Geneva, Switzerland) was used as shown in previous studies [10,24e27]. Ovarian stimulation was achieved with recombinant FSH (rFSH: Gonal-f®, Merck-Serono, Geneva, Switzerland, or Puregone®, Organon, Espanola S.A., Barcelona, Spain) and highly-purified hMG (hp-hMG: Menopur®; Ferring Pharmaceuticals, Geneva, Switzerland), containing 75 IU/ ampule of FSH and LH activity. The selection of the type of gonadotropin or antagonist was individualized according to each patient's characteristics. The starting dose of gonadotropin ranged 150e300 IU, individualized according to the patient's age, baseline FSH, and BMI. Serial folliculometry and assessment of serum E2, P4 and LH levels were monitored every 1e2 days, starting from stimulation day 3e5 and continued until the day of hCG administration (hCG-day) for the final oocyte maturation. A step-down regimen of gonadotropin was applied if the serum E2 concentration reached 300 pg/ml on stimulation day 5. The dosage could be adjusted based on hormonal levels and follicular development. The GnRH antagonist was administered when the follicle reached 12e14 mm. Hormone assessment Serum samples were analyzed using Immulite 2000 reproductive hormone assays (Diagnostic Product Corporation, Siemens, Los Angeles, CA, USA). The sensitivity was 0.1 mIu/ml for FSH; 0.05 mIu/ ml for LH; 15 pg/ml for E2 and 0.1 ng/ml for P4. Intra-assay and inter-assay coefficients of variation were 3.6% and 4.3% for FSH; 4.8% and 10.7% for LH; 6.7% and 9.7% for E2 and 9.7% and 12.2% for P4, respectively. Oocyte retrieval, fertilization and embryo grading A 6500e13,000 IU dose of hCG (Ovidrel®; Merck-Serono, Geneva, Switzerland) according to the patient's age, baseline FSH, and BMI was administered when two or more follicles reached 18 mm in diameter or when per-follicle E2 level reached 100e300 pg/ml [28,29]. Oocyte retrieval was scheduled 34e36 h later and inseminated by ICSI at 39e40 h. The embryos were graded according to the symmetry of the blastomeres and the percentage of anuclear fragments [30]. The surplus embryos were cryopreserved using the Cryotop method [31]. Embryo transfer and luteal support The best morphologically graded embryos from the stimulated cycle were selected for embryo transfer at 2e5 days after oocyte retrieval. In our studies, there were two types of luteal support protocol according to the follicle number. We used 1500 IU hCG (Pregnyl®; Merck Sharp & Dohme, USA) every 3 days and one tube 8% vaginal progesterone gel (Crinone®; Merck-Serono, Geneva, Switzerland) everyday that were started since embryo day 2 for the follicle number 7 on the day of hCG trigger [32]. We used estradiol valerate 2 mg (Estrade®; Synmosa, Taiwan) 4 tablets a day and two tubes of Crinone everyday that were started since embryo day 2 for the follicle number 8 on the day of hCG trigger [7,27]. Serum beta-hCG level was obtained to confirm biochemical pregnancy 16 days after oocyte retrieval. A clinical pregnancy was confirmed with the presence of a fetal cardiac activity at 7 weeks of gestation by transvaginal ultrasonography. The live birth was defined as delevery after 24 weeks of gestation.
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Statistical analysis Values are expressed as mean ± standard deviation or percentage as appropriate. The c2-test, independent t-test or one-way analysis of variance (ANOVA) was applied as appropriate. When comparisons among more than two groups showed significance, post hoc analysis or c2-test was performed for two by two comparisons as appropriate. Statistical analysis was performed using SPSS version 12.0 software. All statistical tests used a two-tailed alpha of 0.05. Results
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similar (37.0 ± 2.7 vs. 36.3 ± 3.3, p ¼ 0.24). The poor responders achieving a pregnancy had higher baseline FSH (p < 0.01), and lower hCG-day E2 (p < 0.01) and P4 (p < 0.01) levels, compared to normal responders achieving a pregnancy. We have found that the numbers of embryos transferred for the pregnant group and the non-pregnant group were not significantly different in the poor responders (1.6 ± 0.8 and 1.5 ± 0.6, respectively, p ¼ 0.46). Those were also similar in the normal responders (2.8 ± 0.8 vs. 2.9 ± 0.9, p ¼ 0.90). After adjusting this confounding factor, we observed that the mean stimulation duration was significant shorter in poor responders achieving a pregnancy than in normal responders achieving a pregnancy (7.8 ± 2.2 vs. 9.2 ± 1.6, p < 0.01).
Baseline characteristics Stimulation duration and clinical pregnancy rate A total of 1481 eligible cycles were analyzed in this study, in which there were 235 (15.9%) poor responders and 1246 (84.1%) normal responders. The demographic characteristics were summarized in Table 1. Overall, the mean age was 37.5 ± 3.7, which ranged from 27 to 51 years old. The mean ET number was 2.6 ± 0.9 and the mean stimulation duration was 9.2 ± 1.7 days. The clinical pregnancy rate and live birth rate were significantly lower in poor responders than in normal responders (clinical pregnancy rate: 29.3% vs. 49.4%, p < 0.001; live birth rate: 17.8% vs. 33.5%, p < 0.001). Clinical parameters in pregnant and non-pregnant patients Clinical parameters in clinically pregnant and non-pregnant women in both poor and normal responders were compared in Table 1. In poor responders, the clinically pregnant patients appeared to have a shorter stimulation duration (7.8 ± 2.2 vs. 8.7 ± 2.1, p ¼ 0.17), but without significance. In normal responders, the clinically pregnant patients and non-pregnant women had similar stimulation duration (9.2 ± 1.6 vs. 9.4 ± 1.6, p ¼ 0.43). Clinical parameters in pregnant women between poor and normal responders We also compared the clinical parameters for pregnant women between poor and normal responders in Table 1. The mean age was
Clinical parameters and IVF outcome among the four groups with different stimulation durations were shown in Table 2 and Fig. 1. In poor responders, the age among these four groups was not significantly different (p ¼ 0.50). Oocyte maturation and fertilization rates among these groups showed no significant differences (p ¼ 0.96 and 0.18, respectively). As the stimulation duration was shorter, the implantation rate (6 days: 36%, 7e8 days: 22%, 9e10 days: 16%, and 11 days: 14%, p ¼ 0.30), clinical pregnancy rate (6 days: 50.0%, 7e8 days: 34.0%, 9e10 days: 23.2%, and 11 days: 22.2%, p ¼ 0.06) and live birth rate (6 days: 33.3%, 7e8 days: 20.0%, 9e10 days: 15.9%, and 11 days: 11.1%, p ¼ 0.18) were higher although without statical significance. In normal responders, the age among four groups was similar (p ¼ 0.15). Patients in longer stimulation duration groups tended to have higher BMI (p < 0.001), higher baseline FSH (p < 0.001), lower baseline E2 (p < 0.001), and higher hCG-day P4 levels (p ¼ 0.1). Oocyte maturation rate was significantly lower at stimulation durations 6 days group (6 days: 67%, 7e8 days: 80%, 9e10 days: 85%, and 11 days: 87%, p ¼ 0.02) and the clinical pregnancy rate appeared lower at stimulation durations 6 days group (6 days: 35.7%, 7e8 days: 53.1%, 9e10 days: 49.1%, and 11 days: 44.7%, p ¼ 0.23). The live birth rate also appeared lower at stimulation durations 6 days group (6 days: 28.6%, 7e8 days: 35.8%, 9e10 days: 33.6%, and 11 days: 29.3%, p ¼ 0.61).
Table 1 The demographic characteristic and IVF outcome in overall patients, poor responders and normal responders. Overall n ¼ 1481
a
Clinically pregnant Case Number Age (years), (range) BMI (kg/m2) Baseline hormone FSH (mIU/ml) E2 (pg/ml) hCG day hormone E2 (pg/ml) P4 (ng/ml) rFSH dosing Initial rFSH (IU) Total rFSH (IU) ET No. Stimulation duration 6 days 7e8 days 9e10 days 11 days Live birth a b c d e
p-valuea,b Normal responder n ¼ 1246 (84.1%)
Poor responder n ¼ 235 (15.9%) b
c
Clinically not pregnant
Clinically pregnant Clinically not pregnant
p-valuec,d p-valuea,c d
<0.01e 0.50
n ¼ 616 (49.4%) n ¼ 630 (50.6%) 36.3 ± 3.3 (27e44) 37.4 ± 3.8 (29e49) 22.23 ± 3.3 21.6 ± 2.7
0.01e 0.22
<0.001e 0.24 0.72
12.1 ± 5.3 34.6 ± 13.6
0.38 0.10
7.9 ± 2.7 37.9 ± 16.0
8.6 ± 4.8 36.7 ± 14.2
0.10 0.48
<0.01e 0.30
538 ± 285 0.58 ± 0.27
589 ± 380 0.56 ± 0.31
0.42 0.61
1900 ± 1351 0.87 ± 0.36
1996 ± 1385 0.89 ± 0.44
0.70 0.46
<0.01e <0.01e
240 ± 62 2050 ± 306 1.6 ± 0.8 7.8 ± 2.2 n ¼ 9 (13.1%) n ¼ 31 (44.9%) n ¼ 19 (27.5%) n ¼ 10 (14.5%) n ¼ 42 (17.8%)
245 ± 53 2057 ± 668 1.5 ± 0.6 8.7 ± 2.1 n ¼ 9 (5.4%) n ¼ 59 (35.5%) n ¼ 63 (38.0%) n ¼ 35 (21.1%)
0.81 0.97 0.46 0.17 0.05 0.18 0.13 0.24
218 ± 39 2004 ± 667 2.8 ± 0.8 9.2 ± 1.6 n ¼ 6 (1.0%) n ¼ 178 (28.9%) n ¼ 348 (56.5%) n ¼ 84 (13.6%) n ¼ 417 (33.5%)
220 ± 46 2002 ± 679 2.9 ± 0.9 9.4 ± 1.6 n ¼ 8 (1.3%) n ¼ 157 (24.9%) n ¼ 361 (57.3%) n ¼ 104 (16.5%)
0.89 0.98 0.90 0.43 0.62 0.10 0.90 0.16
0.06 0.28 <0.01e <0.01e <0.01e <0.01e <0.01e 0.84 <0.001e
n ¼ 1481 n ¼ 69 (29.3%) n ¼ 166 (70.7%) 37.5 ± 3.7 (27e51) 37.0 ± 2.7 (30e43) 40.8 ± 4.7 (28e51) 21.8 ± 3.1 21.4 ± 3.0 22.1 ± 2.4 8.9 ± 4.6 38.4 ± 16.5
13.2 ± 8.6 44.5 ± 29.0
1720 ± 1367 0.81 ± 0.39 224 ± 43 2006 ± 640 2.6 ± 0.9 9.2 ± 1.7 n ¼ 32 (2.2%) n ¼ 425 (28.7%) n ¼ 791 (53.4%) n ¼ 233 (15.7%)
Poor responder with clinically pregnant. Poor responder with clinically not pregnant. Normal responder with clinically pregnant. Normal responder with clinically not pregnant. Statistically significant.
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Table 2 Clinical characteristics and IVF outcome in different stimulation duration groups among poor responders and normal responders. Poor responders (n ¼ 235)
Normal responders (n ¼ 1246)
6 days
7e8days
9e10days
11 days
Case Number
n ¼ 18 (7.6%) 39.0 ± 2.9 (34e43) 22.3 ± 3.1
n ¼ 82 (35.0%) 39.8 ± 4.4 (32e49) 21.9 ± 2.8
n ¼ 45 (19.1%)
Age (years), (range) BMI (kg/m2) Baseline hormone FSH (IU/l) E2 (pg/ml) hCG day hormone E2 (pg/ml) P4 (ng/ml) rFSH dosing Initial rFSH (IU) Total rFSH (IU) ET No. IVF outcome Ovum pick-up No. Maturation rate Fertilization rate Implantation rate Clinical pregnancy rate Live birth rate
n ¼ 90 (38.3%) 39.4 ± 4.5 (30e51) 22.2 ± 2.6
38.5 ± 4.5 (28e43) 22.2 ± 2.7
0.50
12.2 ± 6.6 48.0 ± 21.7
11.8 ± 4.9 39.9 ± 17.3
12.1 ± 5.5 32.4 ± 11.27
460 ± 280 0.29 ± 0.05
569 ± 394 0.46 ± 0.22
217 ± 40 1072 ± 435 1.40 ± 0.5 1.8 ± 0.7 0.87 ± 0.20 0.78 ± 0.24 0.36 ± 0.50 9/18 (50.0%)
a
6/18 (33.3%)
6 days
7e8days
9e10days
11 days
p (ANOVA)
n ¼ 14 (1.1%)
n ¼ 709 (57.0%) 36.7 ± 3.5 (27e49) 22.0 ± 2.9
n ¼ 188 (15.0%) 37.8 ± 3.3 (30e47) 23.4 ± 4.2
0.15
0.96
37.7 ± 4.0 (34e43) 21.4 ± 2.3
n ¼ 335 (26.9%) 37.0 ± 3.9 (27e45) 21.8 ± 2.6
19.8 ± 9.9 23.8 ± 12.6
<0.001a <0.001a
8.3 ± 4.5 58.2 ± 24.8
7.5 ± 2.5 41.9 ± 19.2
8.4 ± 2.8 36.6 ± 12.6
10.0 ± 7.8 33.2 ± 9.7
<0.001a <0.001a
724.± 390 0.69 ± 0.40
424 ± 290 0.55 ± 0.34
0.11 <0.001a
690 ± 336 0.60 ± 0.48
1880 ± 1529 0.79 ± 0.35
2080 ± 1312 0.88 ± 0.37
1852 ± 1224 0.96 ± 0.46
0.14 0.1
238 ± 50 1772 ± 493 1.6 ± 0.6
245 ± 52 2285 ± 625 1.5 ± 0.5
250 ± 50 3075 ± 940 1.6 ± 0.60
0.80 <0.001a 0.39
213 ± 15 1180 ± 105 2.4 ± 0.7
218 ± 22 1608 ± 261 2.7 ± 0.7
228 ± 43 2039 ± 450 2.5 ± 0.8
235 ± 48 3150 ± 870 2.6 ± 0.8
0.02a <0.001a 0.82
2.2 ± 0.7 0.85 ± 0.24 0.75 ± 0.23 0.22 ± 0.42 31/90 (34.0%) 18/90 (20.0%)
2.4 ± 0.6 0.88 ± 0.20 0.68 ± 0.31 0.16 ± 0.61 19/82 (23.2%) 13/82 (15.9%)
2.1 ± 0.7 0.89 ± 0.23 0.78 ± 0.24 0.14 ± 0.46 10/45 (22.2%)
0.02a 0.96 0.18 0.30 0.06
6.3 ± 3.0 0.67 ± 0.23 0.67 ± 0.28 0.15 ± 0.24 5/14 (35.7%)
0.10 0.02a 0.38 0.64 0.23
0.18
4/14 (28.6%)
8.54 ± 3.2 0.85 ± 0.16 0.69 ± 0.14 0.19 ± 0.30 348/709 (49.1%) 238/709 (33.6%)
7.9 ± 3.6 0.87 ± 0.20 0.68 ± 0.22 0.17 ± 0.26 84/188 (44.7%)
5/45 (11.1%)
7.9 ± 3.5 0.80 ± 0.20 0.70 ± 0.18 0.20 ± 0.28 178/335 (53.1%) 120/335 (35.8%)
55/188 (29.3%)
0.61
p (ANOVA)
<0.001a
Statistically significant.
Fig. 1. Clinical characteristics and IVF outcome in different stimulation duration groups among poor responders and normal responders. In poor responders, shortest stimulation duration (6 days) appeared higher clinical pregnancy and live birth rates. In normal responders, shortest stimulation duration (6 days) had lower oocyte maturation, clinical pregnancy and live birth rates.
A significantly lower oocyte maturation rate was found in normal responders with a stimulation duration 6 days than in those with a stimulation duration 7 days (67% vs. 87%, p ¼ 0.005), but the oocyte maturation rates were similar between them in poor responders (6 days: 87% vs. 7 days: 88%, p ¼ 0.76). For the poor responders, the percentages of surplus embryos to be frozen were 0%, 3.3%, 2.4% and 4.4% in the stimulation durations of 6, 7e8, 9e10, and 11 days, respectively. For the normal responders, the percentages of surplus embryos to be frozen were 12.8%, 39.0%, 36.7% and 32.8% in the stimulation duration of 6, 7e8, 9e10, and 11 days, respectively. A significantly lower percentage of cryopreservation was found in normal responders with a stimulation duration 6 days than in those with a stimulation duration 7 days (p < 0.05). The percentages of cryopreservation were not significantly different between them in poor responders (p > 0.05).
Discussion In this study, we demonstrated that in the GnRH antagonist protocol, the duration of gonadotropin stimulation had different impact on oocyte maturity and pregnancy potentials in both poor and normal responders. In poor responders, women who achieved a pregnancy appeared to have a shorter stimulation duration. The role of stimulation duration in IVF cycles of poor responders using GnRH antagonist protocol has never been reported before. Oocyte maturity plays an important role in achieving successful fertilization, subsequent embryo development and pregnancy. It requires sufficient action of LH and FSH on both theca cells and granulosa cells to achieve oocyte maturity and biosynthesize adequate estradiol [33]. The oocyte maturation rate was usually defined by MII oocyte number divided by total oocyte number. It
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represented only nuclear maturation, i.e. resumption and progression of meiosis to MII. In normal responders, the oocyte maturation rate in the 6-days group was significantly lower than other groups, and it increased as the stimulation duration increased (6 days: 67%, 7e8 days: 80%, 9e10 days: 85%, and 11 days: 87%, p ¼ 0.02). It appears that the stimulation duration less than 6 days in COS is probably not sufficient for oocyte maturation in normal responders. Follicular sizes, estradiol and progesterone levels were traditionally employed to determine the timing of triggering final maturation of oocytes [13,14,17,28,29,34]. In our study, the oocyte maturation rate, clinical pregnancy rate, live birth rate, and percentage of cryopreservation for suplus embryos were lower in the 6-days group in normal responders. Therefore, we suggest ovarian response and the stimulation duration may both merit clinical considerations in determining the timing of triggering final oocyte maturation. Different from those in normal responders, oocyte maturation rates in poor responders were similar with different stimulation durations. The implantation and clinical pregnancy rates appeared higher in women with shorter stimulation duration. Shorter proliferative phase seems not to affect the endometrial receptivity. A higher baseline E2 level was also observed to be associated with a shorter stimulation duration (p < 0.001) in poor responders. The higher baseline E2 level may reflect an earlier follicular recruitment initiated by endogenous gonadotropin at time of late luteal and early follicular phase [35,36]. This may lead to a faster follicular growth and a relatively shorter follicular phase during COS. The shorter stimulation duration in poor responders may reflect a better granulosa cell function and oocyte quality, which might contribute a better pregnancy outcome. Besides, the hCG-day P4 level was higher when stimulation duration increased, and the hCG-day P4 level might interfere with endometrial receptivity. Endometrial progesterone receptors could be induced by supra-physiological estradiol level and were significantly up-regulated in stimulated cycles [9]. During COS, a subtle rise in serum progesterone could occur due to increased number of LH receptors and sensitivity of granulosa cells to exogenous gonadotropin [37e39]. Prematurely elevated serum progesterone may cause advanced secretory transformation of endometrium resulting in early closure of the implantation window [14,15,37]. The possible impaired endometrial receptivity was also supported by previous randomized trial that compared fresh and frozenethawed embryo transfer in normal responders [40]. Some recent studies observed the association between a decreased pregnancy rate and elevated hCG-day P4 level (>1.5 ng/ml) in poor responder [37,41], but the stimulation durations were not mentioned in these reports. A longer stimulation duration and a higher hCG-day P4 level was found in the GnRH agonist long protocol than those in the antagonist protocol [37]. Previous studies analyzing the effect of stimulation durations on the pregnancy outcome revealed inconsistent results, in which long GnRH agonist protocol was generally adopted [11,18,19]. Martin et al. reported no significant difference in clinical pregnancy rates among groups with stimulation duration 6e9 days, 10e11 days and 12 days (30.9%, 22.8% and 29.6%, respectively) [18]. Chuang et al. reported a decreased clinical pregnancy rate in cycles with prolonged gonadotropin stimulation 13 days (9 days: 36%, 10e12 days: 37.8%, 13 days: 24.4%, p < 0.01) [19]. Yoldemir et al. showed that the cycles with a stimulation duration 8 days had significantly more poor quality embryos and had a lower clinical pregnancy rate, though there was no significant difference (8 days: 41.1%, 9e10 days: 53.1%, 11 days: 49.7%) [11]. We had tried to divide our patients into three groups (8 days, 9e10 days, and 11 days) as the previous study. However, the finding of different oocyte maturation rates cannot be
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demonstrated. When we divided our patients into 4 groups (6 days, 7e8 days, 9e10 days, and 11 days), the significance of difference was found in normal responders. It appears that the stimulation duration less than 6 days is probably not sufficient for adequate oocyte maturation in normal responders. But, in the poor responders, it appears that the stimulation duration less than 6 days is sufficient for oocyte maturation for patients with earlier recruitment. Therefore, we divided our patients into 4 groups (6 days, 7e8 days, 9e10 days, and 11 days). The effect of stimulation duration in the long GnRH agonist protocol may need further exploration in poor and normal responders. Conclusions Our results demonstrated that in the GnRH antagonist protocol, the duration of gonadotropin stimulation has different impacts on oocyte maturity and pregnancy outcome between poor and normal responders. The patents belonging to the extremely short stimulation duration group (6 days) in normal responders had oocyte maturation rate significantly lower than other groups. This finding implied that it was too early to trigger ovulation in our COS protocol for those patients. It appears that the stimulation duration less than 6 days in COS is probably not sufficient for oocyte maturation in normal responders. Therefore, in addition to follicular sizes and estradiol levels, we may need to concern stimulation duration in determining the timing of triggering final oocyte maturation. In poor responders with early follicular recruitment; however, women with shorter stimulation duration didn't have a lower oocyte maturation rate. Therefore, poor and normal responders should be judged differently during COS with regard to the presence of early follicular recruitment, follicular growth rate, endometrial receptivity, and the stimulation durations. An optimal FSH stimulation duration together with follicular size criteria, serum estradiol and progesterone levels are important clinical parameters in determining timing for hCG to balance between oocyte maturity and endometrial receptivity. In fresh ICSI-ET cycles, stimulation duration appears to have different impacts in both normal and poor responders. It was possible that insufficient stimulation duration resulted in lower oocytes maturation in normal responders, whereas prolonged stimulation duration resulted in less competent oocytes in poor responders. Conflicts of interest statement The authors have declared that no competing interests exist. Financial disclosure The authors received no specific funding for this work. References € €nmezer M, Berker B, Aytaç R, et al. Protocol [1] S¸ükür YE, Ulusoy CO, Ozmen B, So shift from agonist to antagonist or vice versa after an unsuccessful intracytoplasmic sperm injection cycle on the same patient does not improve outcome. Taiwan J Obstet Gynecol 2018;57:417e20. [2] Al-Inany HG, Youssef MA, Aboulghar M, Broekmans F, Sterrenburg M, Smit J, et al. Gonadotrophin-releasing hormone antagonists for assisted reproductive technology. Cochrane Database Syst Rev 2011;5:CD001750. [3] Liang IT, Huang HY, Wu HM, Wang HS, Yu HT, Huang SY, et al. A gonadotropin releasing hormone agonist trigger of ovulation with aggressive luteal phase support for patients at risk of ovarian hyperstimulation syndrome undergoing controlled ovarian hyperstimulation. Taiwan J Obstet Gynecol 2015;54: 583e7. [4] Wang HL, Lai HH, Chuang TH, Shih YW, Huang SC, Lee MJ, et al. A patient friendly corifollitropin alfa protocol without routine pituitary suppression in normal responders. PLoS One 2016;11:e0154123.
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[5] Hwang JL, Chen SU, Chen HJ, Chen HF, Yang YS, Chang CH, et al. Feasibility of corifollitropin alfa/GnRH antagonist protocol combined with GnRH agonist triggering and freeze-all strategy in polycystic ovary syndrome patients. J Formos Med Assoc 2018;117:535e40. [6] Aydogan Mathyk B, Aslan Cetin B, Vardagli D, Zengin E, Sofiyeva N, Irez T, et al. Comparison of antagonist mild and long agonist protocols in terms of follicular fluid total antioxidant capacity. Taiwan J Obstet Gynecol 2018;57:194e9. [7] Wu MY, Chung CH, Pan SP, Jou GC, Chen MJ, Chang CH, et al. Advantages of cumulative pregnancy outcomes in freeze-all strategy in high responders - a case-control matching analysis of a large cohort. J Formos Med Assoc 2018;117:676e84. [8] Mochtar MH, Dutch Ganirelix Study Group. The effect of an individualized GnRH antagonist protocol on folliculogenesis in IVF/ICSI. Hum Reprod 2004;19:1713e8. [9] Papanikolaou EG, Bourgain C, Kolibianakis E, Tournaye H, Devroey P. Steroid receptor expression in late follicular phase endometrium in GnRH antagonist IVF cycles is already altered, indicating initiation of early luteal phase transformation in the absence of secretory changes. Hum Reprod 2005;20:1541e7. [10] Depalo R, Lorusso F, Palmisano M, Bassi E, Totaro I, Vacca M, et al. Follicular growth and oocyte maturation in GnRH agonist and antagonist protocols for in vitro fertilisation and embryo transfer. Gynecol Endocrinol 2009;25: 328e34. [11] Yoldemir T. Does the duration of gonadotropin stimulation affect embryo quality on post-retrieval day 3? Gynecol Endocrinol 2011;27:324e30. [12] Tao T, Robichaud A, Nadeau S, Savoie R, Gallant B, Ouellette RJ. Optimized hormonal stimulation is critical for production of viable embryos and establishment of subsequent implantation. J Assist Reprod Genet 2006;23:23e8. [13] Kolibianakis EM, Bourgain C, Papanikolaou EG, Camus M, Tournaye H, Van Steirteghem AC, et al. Prolongation of follicular phase by delaying hCG administration results in a higher incidence of endometrial advancement on the day of oocyte retrieval in GnRH antagonist cycles. Hum Reprod 2005;20: 2453e6. [14] Huang CC, Lien YR, Chen HF, Chen MJ, Shieh CJ, Yao YL, et al. The duration of pre-ovulatory serum progesterone elevation before hCG administration affects the outcome of IVF/ICSI cycles. Hum Reprod 2012;27:2036e45. [15] Labarta E, Martinez-Conejero JA, Alama P, Horcajadas JA, Pellicer A, Simon C, et al. Endometrial receptivity is affected in women with high circulating progesterone levels at the end of the follicular phase: a functional genomics analysis. Hum Reprod 2011;26:1813e25. [16] Kyrou D, Kolibianakis EM, Fatemi HM, Tarlatzis BC, Tournaye H, Devroey P. Is earlier administration of human chorionic gonadotropin (hCG) associated with the probability of pregnancy in cycles stimulated with recombinant follicle-stimulating hormone and gonadotropin-releasing hormone (GnRH) antagonists? A prospective randomized trial. Fertil Steril 2011;96:1112e5. [17] Mochtar MH, Custers IM, Koks CA, Bernardus RE, Verhoeve HR, Mol BW, et al. Timing oocyte collection in GnRH agonists down-regulated IVF and ICSI cycles: a randomized clinical trial. Hum Reprod 2011;26:1091e6. [18] Martin JR, Mahutte NG, Arici A, Sakkas D. Impact of duration and dose of gonadotrophins on IVF outcomes. Reprod Biomed Online 2006;13:645e50. [19] Chuang M, Zapantis A, Taylor M, Jindal SK, Neal-Perry GS, Lieman HJ, et al. Prolonged gonadotropin stimulation is associated with decreased ART success. J Assist Reprod Genet 2010;27:711e7. [20] Mardesic T, Mannaerts B, Abuzeid M, Levy M, Witjes H, Fauser BC, et al. Short follicular phase of stimulation following corifollitropin alfa or daily recombinant FSH treatment does not compromise clinical outcome: a retrospective analysis of the Engage trial. Reprod Biomed Online 2014;28:462e8. [21] Marci R, Caserta D, Dolo V, Tatone C, Pavan A, Moscarini M. GnRH antagonist in IVF poor-responder patients: results of a randomized trial. Reprod Biomed Online 2005;11:189e93. [22] Pu D, Wu J, Liu J. Comparisons of GnRH antagonist versus GnRH agonist protocol in poor ovarian responders undergoing IVF. Hum Reprod 2011;26: 2742e9. [23] Madani T, Ashrafi M, Yeganeh LM. Comparison of different stimulation protocols efficacy in poor responders undergoing IVF: a retrospective study. Gynecol Endocrinol 2012;28:102e5. [24] Lee TH, Wu MY, Chen HF, Chen MJ, Ho HN, Yang YS. Ovarian response and follicular development for single-dose and multiple-dose protocols for
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Taiwanese Journal of Obstetrics & Gynecology journal homepage: www.tjog-online.com
Original Article
The different impact of stimulation duration on oocyte maturation and pregnancy outcome in fresh cycles with GnRH antagonist protocol in poor responders and normal responders Yu-Chieh Yang a, Yi-Ping Li b, Song-Po Pan a, Kuang-Han Chao a, Chin-Hao Chang c, Jehn-Hsiahn Yang a, Shee-Uan Chen a, * a b c
Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, Taiwan Department of Obstetrics & Gynecology, Shin Kong Memorial Wu Ho-Su Hospital, Taipei, Taiwan Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 14 March 2019
Objective: To study the impact of stimulation duration on intracytoplasmic sperm injection (ICSI) embryo transfer (ET) outcome in poor and normal responders during controlled ovarian stimulation using gonadotropin-releasing hormone (GnRH) antagonist protocol. Materials and methods: This is a retrospective cohort study. There were 1481 women undergoing ICSI-ET cycles. Women with ovum pick-up number 3 were defined as poor responders (n ¼ 235), and those with a number 4 were normal responders (n ¼ 1246). Results: The mean stimulation duration was shorter in poor responders with pregnancy group as compared with normal responders with pregnancy group (7.8 ± 2.2 vs. 9.2 ± 1.6 days, p < 0.01). Poor responders with a shortest stimulation duration (6 days) appeared a higher live birth rate (6 days: 33.3%, 7e8 days: 20.0%, 9e10 days: 15.9%, and 11 days: 11.1%, p ¼ 0.18). Normal responders with a shortest stimulation duration (6 days) appeared a lowest live birth rate (6 days: 28.6%, 7e8 days: 35.8%, 9e10 days: 33.6%, and 11 days: 29.3%, p ¼ 0.61). Oocyte maturation rate was significantly lower at stimulation durations 6 days group (6 days: 67%, 7e8 days: 80%, 9e10 days: 85%, and 11 days: 87%, p ¼ 0.02) in normal responders. Conclusion: In ICSI-ET cycles, stimulation duration appears to have different impact on oocyte maturation, clinical pregnancy rates and live birth rates in both poor and normal responders. © 2019 Taiwan Association of Obstetrics & Gynecology. Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: GnRH antagonist Controlled ovarian stimulation Poor responder Normal responder Stimulation duration
Introduction The long protocol of gonadotropin-releasing hormone (GnRH) agonist in combination with gonadotropin has been traditionally used for controlled ovarian stimulation (COS) in in-vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSIET) treatments [1]. In recent years, the GnRH antagonist protocol becomes increasingly utilized due to its advantage in being patientfriendly and in reduction of ovarian hyperstimulation syndrome (OHSS) [2e7]. An updated meta-analysis comparing the GnRH antagonist protocol and conventional GnRH agonist long protocol
* Corresponding author. Department of Obstetrics and Gynecology, National Taiwan University Hospital, No. 8 Chung-Shan South Road, Taipei, Taiwan. Fax: þ886 2 23116056. E-mail address: [email protected] (S.-U. Chen).
had shown a comparable pregnancy rate and reduction in OHSS [2]. The GnRH antagonist protocol has been reported to have shorter stimulation duration than GnRH agonist protocol [8,9]. However, the effect of gonadotropin stimulation duration on IVF/ICSI outcome in the GnRH antagonist protocol has not been clearly studied. The duration of gonadotropin stimulation is associated with follicular growth, oocyte maturation, oocyte quality and endometrial development. Insufficient period of gonadotropin exposure may lead to nuclear or cytoplasmic immaturity of oocytes [10,11]. On the other hand, prolonged stimulation duration may cause postmaturity of oocytes or apoptosis of granulosa cells and oocytes [12]. It may also result in elevated progesterone (P4) and estradiol (E2) levels and impaired endometrial receptivity [13e15]. Various criteria, including follicular size and estradiol levels, have been
https://doi.org/10.1016/j.tjog.2019.05.007 1028-4559/© 2019 Taiwan Association of Obstetrics & Gynecology. Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
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applied in determining the time of triggering final oocyte maturation [13,16,17]. Only few studies have investigated the correlation between the length of follicular phase and IVF outcome [11,18,19]. The cycles analysed in these reports were mostly GnRH agonist long protocol, and their results were inconclusive. Martin et al. reported no statistically significant difference in clinical pregnancy rates among women with different stimulation durations [18]. Mardesic et al. reported that women with short duration of stimulation of only 6e8 days have the same chance of pregnancy as those with a longer duration of stimulation [20]. Another study by Chuang et al. showed a worse pregnancy outcome with a longer stimulation duration [19]. Due to their different mechanisms and longer gonadotropin stimulation in the long GnRH agonist protocol, these studies might not be applicable to GnRH antagonist cycles. Besides, poor ovarian responders and normal responders may even have different patterns of follicular recruitment and growth rate [10,21e23]. Therefore, they might need to be analyzed separately. In this study, we aim to investigate the impact of stimulation duration of gonadotropin on oocyte maturity, hormonal milieu relating to endometrium, pregnancy rates and live birth rates in the GnRH antagonist cycles. The differences in the effect of stimulation duration on IVF outcomes between poor ovarian responders and normal responders are explored. This study could be helpful in understanding the influence of duration of ovarian stimulation on the IVF outcomes using the GnRH antagonist protocol. Materials and methods Study population This was a retrospective, single-center cohort study, and was approved by the Research Ethics Committee of National Taiwan University Hospital (NTUH). Patients were treated with routine practice of assisted reproductive technologies using the flexible GnRH antagonist protocol [10,24] from January 2009 to June 2017 at NTUH reproductive medicine center. The data were collected from medical records and the computerized database. The inclusion criteria were: eligible indications for COS before ICSI due to male factor using a GnRH-antagonist protocol. Only the first stimulated ICSI cycles without pretreatment were included for analysis in this study. The exclusion criteria were: women with uterine-factor infertility; canceled cycles that did not reach oocyte retrieval; oocyte frozen or oocyte donation cycles; and embryos all frozen due to risk of OHSS. Patient characteristics, including age, body mass index (BMI), and basal hormone levels were recorded. Clinical parameters, including stimulation duration of gonadotropin, gonadotropin dosage, hormone levels at human chorionic gonadotropin (hCG) day, fertilization method, and number of embryos transferred were documented. Other parameters, including number of oocytes retrieved, maturation rate of oocytes, fertilization rate, good embryo rate, implantation rate, biochemical pregnancy rate, clinical pregnancy rate, and live birth rate were analyzed. Study end-points were oocyte maturation rate, clinical pregnancy rate, and live birth rate. Oocyte maturation rate, expressed as metaphase II (MII) oocytes per retrieved oocytes were calculated and analyzed. The patients were categorized according to their ovarian response as poor responders and normal responders. In this study, poor responders were defined as ovum pick-up number 3. To further demonstrate the effect of stimulation duration on each clinical variable, both poor and normal responders were categorized into four groups according to the stimulation durations (6 days, 7e8 days, 9e10 days, and 11 days) to reflect a clinically applicable length of stimulation duration.
Protocols for COS A flexible GnRH antagonist protocol (GnRH antagonist: Orgalutran®, ganirelix; Organon, Dublin, Ireland, or Cetrotide®, cetrorelix acetate; Merck-Serono, Geneva, Switzerland) was used as shown in previous studies [10,24e27]. Ovarian stimulation was achieved with recombinant FSH (rFSH: Gonal-f®, Merck-Serono, Geneva, Switzerland, or Puregone®, Organon, Espanola S.A., Barcelona, Spain) and highly-purified hMG (hp-hMG: Menopur®; Ferring Pharmaceuticals, Geneva, Switzerland), containing 75 IU/ ampule of FSH and LH activity. The selection of the type of gonadotropin or antagonist was individualized according to each patient's characteristics. The starting dose of gonadotropin ranged 150e300 IU, individualized according to the patient's age, baseline FSH, and BMI. Serial folliculometry and assessment of serum E2, P4 and LH levels were monitored every 1e2 days, starting from stimulation day 3e5 and continued until the day of hCG administration (hCG-day) for the final oocyte maturation. A step-down regimen of gonadotropin was applied if the serum E2 concentration reached 300 pg/ml on stimulation day 5. The dosage could be adjusted based on hormonal levels and follicular development. The GnRH antagonist was administered when the follicle reached 12e14 mm. Hormone assessment Serum samples were analyzed using Immulite 2000 reproductive hormone assays (Diagnostic Product Corporation, Siemens, Los Angeles, CA, USA). The sensitivity was 0.1 mIu/ml for FSH; 0.05 mIu/ ml for LH; 15 pg/ml for E2 and 0.1 ng/ml for P4. Intra-assay and inter-assay coefficients of variation were 3.6% and 4.3% for FSH; 4.8% and 10.7% for LH; 6.7% and 9.7% for E2 and 9.7% and 12.2% for P4, respectively. Oocyte retrieval, fertilization and embryo grading A 6500e13,000 IU dose of hCG (Ovidrel®; Merck-Serono, Geneva, Switzerland) according to the patient's age, baseline FSH, and BMI was administered when two or more follicles reached 18 mm in diameter or when per-follicle E2 level reached 100e300 pg/ml [28,29]. Oocyte retrieval was scheduled 34e36 h later and inseminated by ICSI at 39e40 h. The embryos were graded according to the symmetry of the blastomeres and the percentage of anuclear fragments [30]. The surplus embryos were cryopreserved using the Cryotop method [31]. Embryo transfer and luteal support The best morphologically graded embryos from the stimulated cycle were selected for embryo transfer at 2e5 days after oocyte retrieval. In our studies, there were two types of luteal support protocol according to the follicle number. We used 1500 IU hCG (Pregnyl®; Merck Sharp & Dohme, USA) every 3 days and one tube 8% vaginal progesterone gel (Crinone®; Merck-Serono, Geneva, Switzerland) everyday that were started since embryo day 2 for the follicle number 7 on the day of hCG trigger [32]. We used estradiol valerate 2 mg (Estrade®; Synmosa, Taiwan) 4 tablets a day and two tubes of Crinone everyday that were started since embryo day 2 for the follicle number 8 on the day of hCG trigger [7,27]. Serum beta-hCG level was obtained to confirm biochemical pregnancy 16 days after oocyte retrieval. A clinical pregnancy was confirmed with the presence of a fetal cardiac activity at 7 weeks of gestation by transvaginal ultrasonography. The live birth was defined as delevery after 24 weeks of gestation.
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Statistical analysis Values are expressed as mean ± standard deviation or percentage as appropriate. The c2-test, independent t-test or one-way analysis of variance (ANOVA) was applied as appropriate. When comparisons among more than two groups showed significance, post hoc analysis or c2-test was performed for two by two comparisons as appropriate. Statistical analysis was performed using SPSS version 12.0 software. All statistical tests used a two-tailed alpha of 0.05. Results
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similar (37.0 ± 2.7 vs. 36.3 ± 3.3, p ¼ 0.24). The poor responders achieving a pregnancy had higher baseline FSH (p < 0.01), and lower hCG-day E2 (p < 0.01) and P4 (p < 0.01) levels, compared to normal responders achieving a pregnancy. We have found that the numbers of embryos transferred for the pregnant group and the non-pregnant group were not significantly different in the poor responders (1.6 ± 0.8 and 1.5 ± 0.6, respectively, p ¼ 0.46). Those were also similar in the normal responders (2.8 ± 0.8 vs. 2.9 ± 0.9, p ¼ 0.90). After adjusting this confounding factor, we observed that the mean stimulation duration was significant shorter in poor responders achieving a pregnancy than in normal responders achieving a pregnancy (7.8 ± 2.2 vs. 9.2 ± 1.6, p < 0.01).
Baseline characteristics Stimulation duration and clinical pregnancy rate A total of 1481 eligible cycles were analyzed in this study, in which there were 235 (15.9%) poor responders and 1246 (84.1%) normal responders. The demographic characteristics were summarized in Table 1. Overall, the mean age was 37.5 ± 3.7, which ranged from 27 to 51 years old. The mean ET number was 2.6 ± 0.9 and the mean stimulation duration was 9.2 ± 1.7 days. The clinical pregnancy rate and live birth rate were significantly lower in poor responders than in normal responders (clinical pregnancy rate: 29.3% vs. 49.4%, p < 0.001; live birth rate: 17.8% vs. 33.5%, p < 0.001). Clinical parameters in pregnant and non-pregnant patients Clinical parameters in clinically pregnant and non-pregnant women in both poor and normal responders were compared in Table 1. In poor responders, the clinically pregnant patients appeared to have a shorter stimulation duration (7.8 ± 2.2 vs. 8.7 ± 2.1, p ¼ 0.17), but without significance. In normal responders, the clinically pregnant patients and non-pregnant women had similar stimulation duration (9.2 ± 1.6 vs. 9.4 ± 1.6, p ¼ 0.43). Clinical parameters in pregnant women between poor and normal responders We also compared the clinical parameters for pregnant women between poor and normal responders in Table 1. The mean age was
Clinical parameters and IVF outcome among the four groups with different stimulation durations were shown in Table 2 and Fig. 1. In poor responders, the age among these four groups was not significantly different (p ¼ 0.50). Oocyte maturation and fertilization rates among these groups showed no significant differences (p ¼ 0.96 and 0.18, respectively). As the stimulation duration was shorter, the implantation rate (6 days: 36%, 7e8 days: 22%, 9e10 days: 16%, and 11 days: 14%, p ¼ 0.30), clinical pregnancy rate (6 days: 50.0%, 7e8 days: 34.0%, 9e10 days: 23.2%, and 11 days: 22.2%, p ¼ 0.06) and live birth rate (6 days: 33.3%, 7e8 days: 20.0%, 9e10 days: 15.9%, and 11 days: 11.1%, p ¼ 0.18) were higher although without statical significance. In normal responders, the age among four groups was similar (p ¼ 0.15). Patients in longer stimulation duration groups tended to have higher BMI (p < 0.001), higher baseline FSH (p < 0.001), lower baseline E2 (p < 0.001), and higher hCG-day P4 levels (p ¼ 0.1). Oocyte maturation rate was significantly lower at stimulation durations 6 days group (6 days: 67%, 7e8 days: 80%, 9e10 days: 85%, and 11 days: 87%, p ¼ 0.02) and the clinical pregnancy rate appeared lower at stimulation durations 6 days group (6 days: 35.7%, 7e8 days: 53.1%, 9e10 days: 49.1%, and 11 days: 44.7%, p ¼ 0.23). The live birth rate also appeared lower at stimulation durations 6 days group (6 days: 28.6%, 7e8 days: 35.8%, 9e10 days: 33.6%, and 11 days: 29.3%, p ¼ 0.61).
Table 1 The demographic characteristic and IVF outcome in overall patients, poor responders and normal responders. Overall n ¼ 1481
a
Clinically pregnant Case Number Age (years), (range) BMI (kg/m2) Baseline hormone FSH (mIU/ml) E2 (pg/ml) hCG day hormone E2 (pg/ml) P4 (ng/ml) rFSH dosing Initial rFSH (IU) Total rFSH (IU) ET No. Stimulation duration 6 days 7e8 days 9e10 days 11 days Live birth a b c d e
p-valuea,b Normal responder n ¼ 1246 (84.1%)
Poor responder n ¼ 235 (15.9%) b
c
Clinically not pregnant
Clinically pregnant Clinically not pregnant
p-valuec,d p-valuea,c d
<0.01e 0.50
n ¼ 616 (49.4%) n ¼ 630 (50.6%) 36.3 ± 3.3 (27e44) 37.4 ± 3.8 (29e49) 22.23 ± 3.3 21.6 ± 2.7
0.01e 0.22
<0.001e 0.24 0.72
12.1 ± 5.3 34.6 ± 13.6
0.38 0.10
7.9 ± 2.7 37.9 ± 16.0
8.6 ± 4.8 36.7 ± 14.2
0.10 0.48
<0.01e 0.30
538 ± 285 0.58 ± 0.27
589 ± 380 0.56 ± 0.31
0.42 0.61
1900 ± 1351 0.87 ± 0.36
1996 ± 1385 0.89 ± 0.44
0.70 0.46
<0.01e <0.01e
240 ± 62 2050 ± 306 1.6 ± 0.8 7.8 ± 2.2 n ¼ 9 (13.1%) n ¼ 31 (44.9%) n ¼ 19 (27.5%) n ¼ 10 (14.5%) n ¼ 42 (17.8%)
245 ± 53 2057 ± 668 1.5 ± 0.6 8.7 ± 2.1 n ¼ 9 (5.4%) n ¼ 59 (35.5%) n ¼ 63 (38.0%) n ¼ 35 (21.1%)
0.81 0.97 0.46 0.17 0.05 0.18 0.13 0.24
218 ± 39 2004 ± 667 2.8 ± 0.8 9.2 ± 1.6 n ¼ 6 (1.0%) n ¼ 178 (28.9%) n ¼ 348 (56.5%) n ¼ 84 (13.6%) n ¼ 417 (33.5%)
220 ± 46 2002 ± 679 2.9 ± 0.9 9.4 ± 1.6 n ¼ 8 (1.3%) n ¼ 157 (24.9%) n ¼ 361 (57.3%) n ¼ 104 (16.5%)
0.89 0.98 0.90 0.43 0.62 0.10 0.90 0.16
0.06 0.28 <0.01e <0.01e <0.01e <0.01e <0.01e 0.84 <0.001e
n ¼ 1481 n ¼ 69 (29.3%) n ¼ 166 (70.7%) 37.5 ± 3.7 (27e51) 37.0 ± 2.7 (30e43) 40.8 ± 4.7 (28e51) 21.8 ± 3.1 21.4 ± 3.0 22.1 ± 2.4 8.9 ± 4.6 38.4 ± 16.5
13.2 ± 8.6 44.5 ± 29.0
1720 ± 1367 0.81 ± 0.39 224 ± 43 2006 ± 640 2.6 ± 0.9 9.2 ± 1.7 n ¼ 32 (2.2%) n ¼ 425 (28.7%) n ¼ 791 (53.4%) n ¼ 233 (15.7%)
Poor responder with clinically pregnant. Poor responder with clinically not pregnant. Normal responder with clinically pregnant. Normal responder with clinically not pregnant. Statistically significant.
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Table 2 Clinical characteristics and IVF outcome in different stimulation duration groups among poor responders and normal responders. Poor responders (n ¼ 235)
Normal responders (n ¼ 1246)
6 days
7e8days
9e10days
11 days
Case Number
n ¼ 18 (7.6%) 39.0 ± 2.9 (34e43) 22.3 ± 3.1
n ¼ 82 (35.0%) 39.8 ± 4.4 (32e49) 21.9 ± 2.8
n ¼ 45 (19.1%)
Age (years), (range) BMI (kg/m2) Baseline hormone FSH (IU/l) E2 (pg/ml) hCG day hormone E2 (pg/ml) P4 (ng/ml) rFSH dosing Initial rFSH (IU) Total rFSH (IU) ET No. IVF outcome Ovum pick-up No. Maturation rate Fertilization rate Implantation rate Clinical pregnancy rate Live birth rate
n ¼ 90 (38.3%) 39.4 ± 4.5 (30e51) 22.2 ± 2.6
38.5 ± 4.5 (28e43) 22.2 ± 2.7
0.50
12.2 ± 6.6 48.0 ± 21.7
11.8 ± 4.9 39.9 ± 17.3
12.1 ± 5.5 32.4 ± 11.27
460 ± 280 0.29 ± 0.05
569 ± 394 0.46 ± 0.22
217 ± 40 1072 ± 435 1.40 ± 0.5 1.8 ± 0.7 0.87 ± 0.20 0.78 ± 0.24 0.36 ± 0.50 9/18 (50.0%)
a
6/18 (33.3%)
6 days
7e8days
9e10days
11 days
p (ANOVA)
n ¼ 14 (1.1%)
n ¼ 709 (57.0%) 36.7 ± 3.5 (27e49) 22.0 ± 2.9
n ¼ 188 (15.0%) 37.8 ± 3.3 (30e47) 23.4 ± 4.2
0.15
0.96
37.7 ± 4.0 (34e43) 21.4 ± 2.3
n ¼ 335 (26.9%) 37.0 ± 3.9 (27e45) 21.8 ± 2.6
19.8 ± 9.9 23.8 ± 12.6
<0.001a <0.001a
8.3 ± 4.5 58.2 ± 24.8
7.5 ± 2.5 41.9 ± 19.2
8.4 ± 2.8 36.6 ± 12.6
10.0 ± 7.8 33.2 ± 9.7
<0.001a <0.001a
724.± 390 0.69 ± 0.40
424 ± 290 0.55 ± 0.34
0.11 <0.001a
690 ± 336 0.60 ± 0.48
1880 ± 1529 0.79 ± 0.35
2080 ± 1312 0.88 ± 0.37
1852 ± 1224 0.96 ± 0.46
0.14 0.1
238 ± 50 1772 ± 493 1.6 ± 0.6
245 ± 52 2285 ± 625 1.5 ± 0.5
250 ± 50 3075 ± 940 1.6 ± 0.60
0.80 <0.001a 0.39
213 ± 15 1180 ± 105 2.4 ± 0.7
218 ± 22 1608 ± 261 2.7 ± 0.7
228 ± 43 2039 ± 450 2.5 ± 0.8
235 ± 48 3150 ± 870 2.6 ± 0.8
0.02a <0.001a 0.82
2.2 ± 0.7 0.85 ± 0.24 0.75 ± 0.23 0.22 ± 0.42 31/90 (34.0%) 18/90 (20.0%)
2.4 ± 0.6 0.88 ± 0.20 0.68 ± 0.31 0.16 ± 0.61 19/82 (23.2%) 13/82 (15.9%)
2.1 ± 0.7 0.89 ± 0.23 0.78 ± 0.24 0.14 ± 0.46 10/45 (22.2%)
0.02a 0.96 0.18 0.30 0.06
6.3 ± 3.0 0.67 ± 0.23 0.67 ± 0.28 0.15 ± 0.24 5/14 (35.7%)
0.10 0.02a 0.38 0.64 0.23
0.18
4/14 (28.6%)
8.54 ± 3.2 0.85 ± 0.16 0.69 ± 0.14 0.19 ± 0.30 348/709 (49.1%) 238/709 (33.6%)
7.9 ± 3.6 0.87 ± 0.20 0.68 ± 0.22 0.17 ± 0.26 84/188 (44.7%)
5/45 (11.1%)
7.9 ± 3.5 0.80 ± 0.20 0.70 ± 0.18 0.20 ± 0.28 178/335 (53.1%) 120/335 (35.8%)
55/188 (29.3%)
0.61
p (ANOVA)
<0.001a
Statistically significant.
Fig. 1. Clinical characteristics and IVF outcome in different stimulation duration groups among poor responders and normal responders. In poor responders, shortest stimulation duration (6 days) appeared higher clinical pregnancy and live birth rates. In normal responders, shortest stimulation duration (6 days) had lower oocyte maturation, clinical pregnancy and live birth rates.
A significantly lower oocyte maturation rate was found in normal responders with a stimulation duration 6 days than in those with a stimulation duration 7 days (67% vs. 87%, p ¼ 0.005), but the oocyte maturation rates were similar between them in poor responders (6 days: 87% vs. 7 days: 88%, p ¼ 0.76). For the poor responders, the percentages of surplus embryos to be frozen were 0%, 3.3%, 2.4% and 4.4% in the stimulation durations of 6, 7e8, 9e10, and 11 days, respectively. For the normal responders, the percentages of surplus embryos to be frozen were 12.8%, 39.0%, 36.7% and 32.8% in the stimulation duration of 6, 7e8, 9e10, and 11 days, respectively. A significantly lower percentage of cryopreservation was found in normal responders with a stimulation duration 6 days than in those with a stimulation duration 7 days (p < 0.05). The percentages of cryopreservation were not significantly different between them in poor responders (p > 0.05).
Discussion In this study, we demonstrated that in the GnRH antagonist protocol, the duration of gonadotropin stimulation had different impact on oocyte maturity and pregnancy potentials in both poor and normal responders. In poor responders, women who achieved a pregnancy appeared to have a shorter stimulation duration. The role of stimulation duration in IVF cycles of poor responders using GnRH antagonist protocol has never been reported before. Oocyte maturity plays an important role in achieving successful fertilization, subsequent embryo development and pregnancy. It requires sufficient action of LH and FSH on both theca cells and granulosa cells to achieve oocyte maturity and biosynthesize adequate estradiol [33]. The oocyte maturation rate was usually defined by MII oocyte number divided by total oocyte number. It
Y.-C. Yang et al. / Taiwanese Journal of Obstetrics & Gynecology 58 (2019) 471e476
represented only nuclear maturation, i.e. resumption and progression of meiosis to MII. In normal responders, the oocyte maturation rate in the 6-days group was significantly lower than other groups, and it increased as the stimulation duration increased (6 days: 67%, 7e8 days: 80%, 9e10 days: 85%, and 11 days: 87%, p ¼ 0.02). It appears that the stimulation duration less than 6 days in COS is probably not sufficient for oocyte maturation in normal responders. Follicular sizes, estradiol and progesterone levels were traditionally employed to determine the timing of triggering final maturation of oocytes [13,14,17,28,29,34]. In our study, the oocyte maturation rate, clinical pregnancy rate, live birth rate, and percentage of cryopreservation for suplus embryos were lower in the 6-days group in normal responders. Therefore, we suggest ovarian response and the stimulation duration may both merit clinical considerations in determining the timing of triggering final oocyte maturation. Different from those in normal responders, oocyte maturation rates in poor responders were similar with different stimulation durations. The implantation and clinical pregnancy rates appeared higher in women with shorter stimulation duration. Shorter proliferative phase seems not to affect the endometrial receptivity. A higher baseline E2 level was also observed to be associated with a shorter stimulation duration (p < 0.001) in poor responders. The higher baseline E2 level may reflect an earlier follicular recruitment initiated by endogenous gonadotropin at time of late luteal and early follicular phase [35,36]. This may lead to a faster follicular growth and a relatively shorter follicular phase during COS. The shorter stimulation duration in poor responders may reflect a better granulosa cell function and oocyte quality, which might contribute a better pregnancy outcome. Besides, the hCG-day P4 level was higher when stimulation duration increased, and the hCG-day P4 level might interfere with endometrial receptivity. Endometrial progesterone receptors could be induced by supra-physiological estradiol level and were significantly up-regulated in stimulated cycles [9]. During COS, a subtle rise in serum progesterone could occur due to increased number of LH receptors and sensitivity of granulosa cells to exogenous gonadotropin [37e39]. Prematurely elevated serum progesterone may cause advanced secretory transformation of endometrium resulting in early closure of the implantation window [14,15,37]. The possible impaired endometrial receptivity was also supported by previous randomized trial that compared fresh and frozenethawed embryo transfer in normal responders [40]. Some recent studies observed the association between a decreased pregnancy rate and elevated hCG-day P4 level (>1.5 ng/ml) in poor responder [37,41], but the stimulation durations were not mentioned in these reports. A longer stimulation duration and a higher hCG-day P4 level was found in the GnRH agonist long protocol than those in the antagonist protocol [37]. Previous studies analyzing the effect of stimulation durations on the pregnancy outcome revealed inconsistent results, in which long GnRH agonist protocol was generally adopted [11,18,19]. Martin et al. reported no significant difference in clinical pregnancy rates among groups with stimulation duration 6e9 days, 10e11 days and 12 days (30.9%, 22.8% and 29.6%, respectively) [18]. Chuang et al. reported a decreased clinical pregnancy rate in cycles with prolonged gonadotropin stimulation 13 days (9 days: 36%, 10e12 days: 37.8%, 13 days: 24.4%, p < 0.01) [19]. Yoldemir et al. showed that the cycles with a stimulation duration 8 days had significantly more poor quality embryos and had a lower clinical pregnancy rate, though there was no significant difference (8 days: 41.1%, 9e10 days: 53.1%, 11 days: 49.7%) [11]. We had tried to divide our patients into three groups (8 days, 9e10 days, and 11 days) as the previous study. However, the finding of different oocyte maturation rates cannot be
475
demonstrated. When we divided our patients into 4 groups (6 days, 7e8 days, 9e10 days, and 11 days), the significance of difference was found in normal responders. It appears that the stimulation duration less than 6 days is probably not sufficient for adequate oocyte maturation in normal responders. But, in the poor responders, it appears that the stimulation duration less than 6 days is sufficient for oocyte maturation for patients with earlier recruitment. Therefore, we divided our patients into 4 groups (6 days, 7e8 days, 9e10 days, and 11 days). The effect of stimulation duration in the long GnRH agonist protocol may need further exploration in poor and normal responders. Conclusions Our results demonstrated that in the GnRH antagonist protocol, the duration of gonadotropin stimulation has different impacts on oocyte maturity and pregnancy outcome between poor and normal responders. The patents belonging to the extremely short stimulation duration group (6 days) in normal responders had oocyte maturation rate significantly lower than other groups. This finding implied that it was too early to trigger ovulation in our COS protocol for those patients. It appears that the stimulation duration less than 6 days in COS is probably not sufficient for oocyte maturation in normal responders. Therefore, in addition to follicular sizes and estradiol levels, we may need to concern stimulation duration in determining the timing of triggering final oocyte maturation. In poor responders with early follicular recruitment; however, women with shorter stimulation duration didn't have a lower oocyte maturation rate. Therefore, poor and normal responders should be judged differently during COS with regard to the presence of early follicular recruitment, follicular growth rate, endometrial receptivity, and the stimulation durations. An optimal FSH stimulation duration together with follicular size criteria, serum estradiol and progesterone levels are important clinical parameters in determining timing for hCG to balance between oocyte maturity and endometrial receptivity. In fresh ICSI-ET cycles, stimulation duration appears to have different impacts in both normal and poor responders. It was possible that insufficient stimulation duration resulted in lower oocytes maturation in normal responders, whereas prolonged stimulation duration resulted in less competent oocytes in poor responders. Conflicts of interest statement The authors have declared that no competing interests exist. Financial disclosure The authors received no specific funding for this work. References € €nmezer M, Berker B, Aytaç R, et al. Protocol [1] S¸ükür YE, Ulusoy CO, Ozmen B, So shift from agonist to antagonist or vice versa after an unsuccessful intracytoplasmic sperm injection cycle on the same patient does not improve outcome. Taiwan J Obstet Gynecol 2018;57:417e20. [2] Al-Inany HG, Youssef MA, Aboulghar M, Broekmans F, Sterrenburg M, Smit J, et al. Gonadotrophin-releasing hormone antagonists for assisted reproductive technology. Cochrane Database Syst Rev 2011;5:CD001750. [3] Liang IT, Huang HY, Wu HM, Wang HS, Yu HT, Huang SY, et al. A gonadotropin releasing hormone agonist trigger of ovulation with aggressive luteal phase support for patients at risk of ovarian hyperstimulation syndrome undergoing controlled ovarian hyperstimulation. Taiwan J Obstet Gynecol 2015;54: 583e7. [4] Wang HL, Lai HH, Chuang TH, Shih YW, Huang SC, Lee MJ, et al. A patient friendly corifollitropin alfa protocol without routine pituitary suppression in normal responders. PLoS One 2016;11:e0154123.
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