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Clomiphene citrate in LH surge suppression for women undergoing ICSI
Middle East Fertility Society Journal xxx (2018) xxx–xxx
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Original Article
Clomiphene citrate in LH surge suppression for women undergoing ICSI Nermeen Mohamad Shams-Eldeen ⇑, Hesham Mahmoud Shalan, Reda Abdel-Hady Hemida, Abdel-Gawad Elmetwally Department of Obstetrics and Gynecology, Mansoura University, Mansoura, Dakahlia, Egypt
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Article history: Received 29 December 2017 Revised 9 January 2018 Accepted 12 January 2018 Available online xxxx Keywords: Clomiphene COS LH surge Premature luteinization
a b s t r a c t Objective: To evaluate the effect of adding clomiphene citrate (CC) in the mid-to-late follicular phase as an adjuvant to gonadotropins to suppress luteinizing hormone (LH) surge in women undergoing intracytoplasmic sperm injection (ICSI). Methods: Prospective non-randomized study of 108 women undergoing ICSI and subjected to ovarian stimulation with gonadotropins with addition of CC (50 mg 3 times per day) when a leading follicle reached 14 mm in diameter and continued till the day of human chorionic gonadotropin (HCG) administration. Women subjected to controlled ovarian stimulation (COS) with the gonadotropin-CC protocol (n = 50) were compared with a group of women were to COS with the flexible gonadotropin releasing hormone antagonist (GnRH-ant) protocol (n = 58). Results: Serum LH level on day HCG administration was significantly higher in CC group than in GnRHant group and the incidence of LH surge was higher in CC group than in GnRH-ant group (10% vs 3.4%, respectively) but without statistically significant difference (P = .246). The oocyte maturation and fertilization rates, the biochemical and clinical pregnancy rates, the implantation rate and the ongoing pregnancy rate were comparable in both groups. Conclusion: Adding CC in the mid-to-late follicular phase as an adjuvant to gonadotropins represents a less costly COS which is effective in eliminating the occurrence of premature LH surge without compromising the cycle outcomes in women undergoing ICSI. Ó 2018 Middle East Fertility Society. Production and hosting 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/).
1. Introduction The field of in vitro fertilization (IVF) has grown since 1978 after the birth of Louise Brown and its success has improved over the past 30 years, and about 5 million babies have been born by 2013 worldwide after IVF treatment, and live birth rates per IVF cycle have noticeably increased, particularly for patients younger than 35 years [1]. The need for assisted reproduction techniques (ARTs) is greater than ever, since up to 10% of couples may suffer from some form of infertility and it aims to provide safe and efficient service to increase rate of pregnancy and live birth [2]. The introduction of gonadotropins for ovarian stimulation made a great improvement in IVF success but faced major challenge in
Peer review under responsibility of Middle East Fertility Society. ⇑ Corresponding author at: Department of Obstetrics and Gynecology, Mansoura University Hospitals, Elgomhouria St., Mansoura City 35111, Dakahlia, Egypt. E-mail address: [email protected] (N.M. Shams-Eldeen).
the occurrence of a premature luteinizing hormone (LH) surge and consequent lutinization before ovarian follicle maturation that associates poor oocyte quality, decreased fertilization and implantation rates [3]. Concomitant administration of a gonadotropin releasing hormone (GnRH) agonist or antagonist through controlled ovarian stimulation (COS) protocols, is used to prevent a premature LH surge, and final oocyte maturation and ovulation is typically triggered with a bolus of GnRH agonist, human chorionic gonadotropin (HCG), or both to improve the response, and obtain good quality oocytes and embryos, with maximizing implantation and pregnancy rates [4]. Clomiphene citrate (CC) has been the most widely used for fertility enhancement for 4 decades. It acts as a selective estrogen receptor modulator, as competitive inhibitor of estrogen binding to estrogen receptors. Most evidence suggested primary site of action on hypothalamic estrogen receptors blocking the negative feedback effect of circulating endogenous estrogen resulting in increased plasma level of LH and follicle stimulating hormone (FSH) [5].
https://doi.org/10.1016/j.mefs.2018.01.005 1110-5690/Ó 2018 Middle East Fertility Society. Production and hosting 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/).
Please cite this article in press as: N.M. Shams-Eldeen et al., Clomiphene citrate in LH surge suppression for women undergoing ICSI, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.01.005
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The standard therapy in the past has been the combination of CC treatment in the early follicular phase and subsequent, overlapping gonadotropin stimulation to decrease the amount of required gonadotropins and the costs. In addition, the gonadotropins counteracts the detrimental effects of the CC to the endometrium. One of the major disadvantages is the high occurrences of premature LH surge and consequent lutinization before ovarian follicle maturation and therefore results in the high cancellation rate [6]. Al-Inany et al. [7] showed the beneficial effects of CC during ovarian stimulation with gonadotropins to prevent a premature LH surge in intrauterine insemination (IUI). The rationale is that its antiestrogenic effect may suppress premature LH rise while maintaining a positive influence on ovarian follicle development. In another study, gonadotropins in combination with CC in midto-late follicular phase was compared to short protocol of GnRH agonist-gonadotropins. The gonadotropins-CC protocol resulted in the same pregnancy outcome as short protocol. The novel protocol may take the advantage of eliminating the occurrence of a premature endogenous LH surge [8]. Recently, Bhandari et al. [9] concluded in a pilot study the feasibility of use of continuous CC beyond the usual 5 days with gonadotropins as an effective tool to prevent premature LH surge without compromising oocyte yield. The paucity of studies evaluating efficacy of CC as a tool to prevent premature LH surge during COS pushed us to conduct this study to evaluate the effect of adding CC in the mid-to-late follicular phase as an adjuvant to gonadotropins to suppress LH surge in women undergoing intracytoplasmic sperm injection (ICSI). 2. Method This was a prospective non-randomized study conducted during the period from January 2014 through December 2015 in Mansoura University Fertility Care Unit (FCU), Egypt. The study was approved by the Mansoura Medical Research Ethics committee (code No. MD/28). The participants of this study were chosen from the couples recruited for management of infertility for different causes by ICSI. A written informed consent was taken from each couple selected to participate in the study. The exclusion criteria were: (1) age < 18 or > 38 years; (2) basal serum FSH > 10 IU/L; (3) PCOS; (4) sever endometriosis; or (5) abnormal uterine cavity. 2.1. Controlled ovarian stimulation (COS) protocol In women in the CC group (n = 50), ovarian stimulation using gonadotropin preparation was commenced on day 3 of the stimulation cycle after performing transvaginal sonography (TVS) scan to confirm absence of ovarian cysts. The gonadotropins were given daily by deep intramuscular injection and the starting dose and type depended on the age of the woman, baseline FSH levels, body mass index (BMI) and previous trials. The TVS scan was performed regularly for monitoring follicular development (folliculometry); starting from day 8 of the cycle and repeated every 2–3 days. The dose and type of gonadotropins were modulated according to ovarian response. The CC administration (ClomidÒ, Clomiphene Citrate 50 mg, Marion Merrell, Lebanon; Sanofi-Aventis, Egypt) was started at a dose of 50 mg 3 times per day when a leading follicle reached 14 mm in diameter and continued till the day of HCG administration. Serum LH concentration was measured at day of HCG injection. A control group (n = 58) was selected from women who underwent COS through GnRH antagonist (GnRH-ant) flexible protocol, matched for age and BMI. In these women, ovarian stimulation using gonadotropin preparation was commenced on day 3 of the stimulation cycle after performing TVS scan to confirm absence
of ovarian cysts. The gonadotropins were given daily by deep intramuscular injection and the starting dose and type depended on the age of the woman, baseline FSH levels, BMI and previous trials. TVS scan was performed regularly for monitoring follicular development (folliculometry); starting from day 8 of the cycle and repeated every 2–3 days. The dose and type of gonadotropins were modulated according to ovarian response. The GnRH-ant preparation (CetrotideÒ, Cetrorelix 0.25 mg, Merck Serono, Darmstadt, Germany) was administered subcutaneously in a dose of 0.25 mg/day when a leading follicle reaches 14 mm in diameter and continued till the day of HCG administration. In both groups, if poor ovarian response (< 4 follicles not reaching 18 mm correlated with serum E2 level < 400 pg/ml) was detected after 11–12 days of ovarian stimulation, the couple were informed by the clinicians about the chances of success and the treatment options (to continue treatment, to have intrauterine insemination, whenever indicated, or to be cancelled), and the decision of management depended essentially on the choice of each couple. The cycle was also cancelled when there is a high risk for OHSS. When the leading follicles reach diameter of 18 mm, final oocyte maturation was induced by intramuscular administration of 10,000 IU of HCG. 2.2. IVF/ICSI protocol On the day of HCG injection in both groups, serum LH and serum progesterone levels were assayed as indicators of premature luteinization. After HCG injection by 34–36 h, oocyte retrieval was performed through transvaginal aspiration of follicles under TVS guidance followed by endometrial preparation for embryo transfer (ET). Transfer of embryos was performed transcervically under ultrasound guidance. Luteal phase support was continued by the same regimen which was started on the day of oocytes retrieval until 2 weeks after ET. 2.3. Outcome measures The primary outcome measures of this study were the clinical pregnancy rate and incidence of LH surge. The secondary outcome measures were the ongoing pregnancy rate, implantation rate and Incidence of early OHSS (within 9 days of HCG injection). Clinical pregnancy was defined as presence of at least one intrauterine gestational sac with fetal pole and cardiac activity on TVS scan at 4–6 weeks after embryo transfer (ET). Clinical pregnancy rate was calculated by dividing the number of clinical pregnancies by the number of ET procedures. LH surge was defined as combination of both premature LH rise ( 10 mIU/ml) and premature progesterone rise (>0.8 ng/ml) [10]. Ongoing pregnancy was defined as pregnancy that progressed beyond the critical first trimester (12 weeks gestational age). Implantation rate was calculated by dividing the number of gestational sacs on TVS scan at 4–6 weeks after ET by the number of transferred embryos. Diagnosis and classification of cases of OHSS was reached through clinical picture as well as laboratory results [11]. 2.4. Sample size calculation The sample size was calculated using the computer statistical software G⁄Power 3.1.9.2 (Fisher’s exact test, two-tailed significance, alpha error probability = 0.05, power = 80%, allocation ratio for groups = 1). A sample size of at least 88 women (44 per group) is needed to detect 20% difference between both groups. The estimation of the sample size was based on the previously reported 2% incidence of premature LH surge in flexible GnRH antagonist IVF/ICSI cycle [12], and the reported 22–28% incidence of premature LH surge in clomiphene citrate / gonadotropin IVF/ICSI cycles
Please cite this article in press as: N.M. Shams-Eldeen et al., Clomiphene citrate in LH surge suppression for women undergoing ICSI, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.01.005
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with or without use of GnRH analogues [13,14]. For examination of data from at least 44 patients in each group in the final analysis, it is appropriate to enroll 50 patients in each group. 2.5. Statistical analysis Continuous variables were presented as mean ± standard deviation while categorical variables were presented as frequencies and percentages. The IBMÒ SPSSÒ Statistics, version 20.0 for Windows was used for statistical analysis. P values .05 were considered statistically significant. 3. Results A total of 108 women were included in the study. In 50 women, gonadotropin-CC protocol was used for COS (CC group) and in 58 women, the GnRH-ant flexible protocol was used for COS (GnRHant group). Demographic and hormonal characteristics of both groups are shown in Table 1. The total gonadotropin dose, number of stimulation days and serum E2 level on day HCG administration were comparable in both CC and GnRH-ant groups. Serum LH level on day HCG administration was significantly higher in CC group than in GnRH-ant group. Serum progesterone level on day HCG administration was also higher in CC group than in GnRH-ant group but without statistically significant difference. Endometrial thickness on day HCG administration was significantly lower in CC group than in GnRH-ant group. The incidence of LH surge and the incidence of early OHSS were higher in CC group than in GnRH-ant group but without statistically significant difference (Table 2). The number of cases with cycle cancellation (before oocyte retrieval) and ET cancellation and the causes of these cancellations in both groups are shown in the patient flow (Fig. 1). Although the number of collected oocytes significantly higher in CC group than in GnRH-ant group, oocyte maturation and fertilization rates were comparable in both groups, but the number of 8-cell stage embryos
Table 1 Demographic and hormonal characteristics of the study groups. CC group (n = 50)
GnRH-ant group (n = 58)
P value
Age (years)* BMI (kg/m2)* Duration of infertility (years)*
30.12 ± 5.37 27.49 ± 3.86 5.36 ± 3.08
31.59 ± 4.62 28.09 ± 4.06 6.57 ± 3.97
.104 .378 .135
Type of infertilityy Primary Secondary Previous IVF/ICSIy
33/50 (66.0%) 17/50 (34.0%) 12/50 (24.0%)
39/58 (67.2%) 19/58 (32.8%) 14/58 (24.1%)
.891
Cause of infertilityy Male factor Tubal factor Endometriosis Unexplained Combined Basal serum FSH (mIU/ml)* Basal serum LH (mIU/ml)* Serum prolactin (ng/ml)* Serum TSH (uIU/ml)*
24/50 (48.0%) 19/50 (38.0%) 7/50 (14.0%) 9/50 (18.0%) 9/50 (18.0%) 6.75 ± 1.71 5.51 ± 2.19 12.26 ± 5.44 1.76 ± 0.69
29/58 (50.0%) 27/58 (46.6%) 7/58 (12.1%) 10/58 (17.2%) 11/58 (19.0%) 6.56 ± 1.47 5.40 ± 2.19 11.91 ± 5.14 1.73 ± 0.70
.836 .370 .766 .918 .898 .500 .878 .912 .784
.987
BMI, body mass index. IVF, in vitro fertilization. ICSI, intracytoplasmic sperm injection. FSH, follicle stimulating hormone. TSH, thyroid stimulating hormone. LH, luteinizing hormone. * Expressed as mean ± SD and P value was calculated by the Mann-Whitney U test. y Expressed as frequency and percentage and P value was calculated by the ChiSquare test.
Table 2 COS characteristics of the study groups.
Total FSH dose (IU)* Stimulation days* Serum E2 on day of HCG (pg/ml)* Serum LH on day of HCG (mIU/ml)* Serum progesterone on day of HCG (ng/ml)* Occurrence of LH surgey Endometrial thickness on day of HCG (mm)* Early OHSSy
CC group (n = 50)
GnRH-ant group (n = 58)
P value
2777 ± 578 9.0 ± 1.2 1982 ± 922 3.99 ± 2.44 0.76 ± 0.59
2820 ± 481 8.9 ± 1.1 1903 ± 849 2.30 ± 1.67 0.57 ± 0.39
.807 .703 .675 <.001 .142
5/50 (10.0%) 8.4 ± 1.2
2/58 (3.4%) 9.1 ± 1.0
.246 .002
3/50 (6.0%)
2/58 (3.4%)
.661
COS, controlled ovarian stimulation. E2, estradiol. HCG, human chorionic gonadotropin. FSH, follicle stimulating hormone. LH, luteinizing hormone. * Expressed as mean ± SD and P value was calculated by the Mann-Whitney U test. y Expressed as frequency and percentage and P value was calculated by the Fisher’s exact test.
was significantly higher in CC group than in GnRH-ant group. The number of transferred embryos and the day of ET were comparable in both groups. Also, the biochemical and clinical pregnancy rates, the implantation rate and the ongoing pregnancy rate were similar in both groups (Table 3).
4. Discussion A great improvement in the success of IVF/ICSI was obtained by the use of gonadotropins for ovarian stimulation but this was associated with increased incidence of premature luteinization and poor cycle outcomes owing to premature rise in LH; therefore, the COS protocols were introduced to prevent occurrence premature LH surge and subsequently, improve the cycle outcomes [3]. The CC is considered a first-line therapy for induction of ovulation owing to low cost, oral administration, and minimal adverse effects which represent advantages over injectable medications [15]. So, use of CC is in line with the recent trend toward ‘‘patient-friendly IVF” which is composed of a mixture of four criteria: cost effectiveness, equity of access, minimal risk for mother and child and minimal load for patients [8,16]. The CC used continuously from day 1 or 2 of the menstrual cycle to block estrogen receptors sites at the hypothalamus and pituitary. The CC was used in this way to imitate the action of gonadotropin analogues and prevents an LH surge due to rising estrogen levels, a result of follicular development [9]. CC has a half-life of 24 h or less, and its discontinuation, as in soft stimulation, can cause the initiation of LH surge. This problem can be solved by continuing CC till the day of trigger [17]. Therefore, we conducted this study to evaluate the efficacy of a simplified COS protocol, in which CC was used as an alternative to GnRH antagonist in order to prevent LH surge without affecting the IVF/ICSI cycle outcomes. The CC was administered in the same dose given in the study of Al-Inany et al. [7] due to similar population of their study and our study and also, the accurate dose was not determined from other previous studies. It appears that the antiestrogenic properties of CC did not affect the stimulatory effect of gonadotropins. This was obvious by the fact that the mean E2 levels were even higher in the CC group as was the number of mature follicles in our study group. It was a matter of concern by Singh et al. [17] that the antiestrogenic effect of CC on the genital tract with its prolonged use might alter the
Please cite this article in press as: N.M. Shams-Eldeen et al., Clomiphene citrate in LH surge suppression for women undergoing ICSI, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.01.005
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Fig. 1. Patient flow chart.
Table 3 Outcomes in cases underwent oocyte retrieval and ET in the study groups.
No. of oocytes retrieved* Oocyte maturation rate (%)* Fertilization rate (%)* No. of 8-cell stage embryos* No. 1 2 3 4 5
of transferred embryosy embryo embryos embryos embryos embryos
Day of ETy Day 2 Day 3 Day 4 Day 5 Biochemical pregnancyà Clinical pregnancyà Implantation rate (%)à Ongoing pregnancyà
CC group (n = 44)
GnRH-ant group (n = 53)
P value
11.1 ± 6.6 83.5 ± 15.0 70.6 ± 19.8 6.2 ± 3.9
8.4 ± 5.2 80.5 ± 22.0 72.5 ± 24.1 4.3 ± 3.0
.033 .953 .444 .009
3/44 (6.8%) 6/44 (13.6%) 23/44 (52.3%) 10/44 (22.7%) 2/44 (4.5%)
9/53 (17.0%) 6/53 (11.3%) 24/53 (45.3%) 10/53 (18.9%) 4/53 (7.5%)
.215 .765 .544 .802 .686
2/44 (4.5%) 28/44 (63.6%) 12/44 (27.3%) 2/44 (4.5%) 16/44 (36.4%) 14/44 (31.8%) 22/134 (16.4%) 12/14 (85.7%)
3/53 (5.7%) 41/53 (77.4%) 7/53 (13.2%) 2/53 (3.8%) 19/53 (35.8%) 16/53 (30.2%) 27/153 (17.6%) 14/16 (87.5%)
1.000 .178 .122 1.000 .958 .863 .782 .886
ET, embryo transfer. OHSS, ovarian hyperstimulation syndrome. * Expressed as mean ± SD and P value was calculated by the Mann-Whitney U test. y Expressed as frequency and percentage and P value was calculated by the Fisher’s exact test. à Expressed as frequency and percentage and P value was calculated by the Chi-Square test.
endometrial receptivity as their protocol used in oocyte donors and when sequential transfer is planned. Although it was statistically significant, there was slight difference (0.6 mm) in the mean endometrial thickness between the CC group and GnRH-ant group in our study. However, the endometrial thickness obtained in our study was higher than that was obtained by Al-Inany et al. [7]. This difference may be attributed to higher doses of gonadotropins which counteract the deleterious effect of CC on the endometrium. On the other hand, the mean endometrial thickness in our study was lower than that was obtained by Kang et al. [8] (nearly 11 mm in both groups). The mean serum LH level on the HCG injection day was significantly higher with the CC protocol in our study, but not to the
extent of occurrence of LH surge (3.99 mIU/ml). This level is lower than that was obtained by Bhandari et al. [9] (6.68 mIU/ml), but higher than that was obtained by Kang et al. [8] (2.51 mIU/ml). These contradictory results could be explained by the lower CC dose (50 gm/d) used by Bhandari et al. [9], and the lower total gonadotropin dose (2033 ± 555 IU) given in the study of Kang et al. [8]. Also, in contrast to our study, Kang et al. al [8] had found significant lower LH level in the CC protocol group than in the short protocol group (2.51 vs 3.64 mIU/ml). This could be explained by the fact that short GnRHa protocol has initial flare up effect but the GnRH antagonist and CC-gonadotropin protocols do not have this effect. Singh et al. [17] have used CC in their study from the beginning of stimulation till the day of trigger in oocyte donors and compared it with the other donors, receiving GnRH-ant flexible protocol. Following the same idea, but we started before publishing of the Singh et al. [17] results, seemed good to allow us for comparing new protocol with the old established one using GnRH-ant in flexible manner (when follicle reaches >14 mm), and from the physiological point that CC when given with high serum E2 will benefit its effect in prevention of LH surge, that was a novel trend and need to be investigated further. The mechanism of triggering LH surge in the hypothalamus is not very clear and it has been reported that LH surge occurs in nearly 20–25% of CC and/or gonadotropin cycles [18]. The extended use of CC in a dose of 100 mg for 8 days (from cycle day 3 to 10) was by applied by Matsaseng et al. [19] to prevent LH surge and thus avoid the use of GnRH analogues and reduce the cost; however, the rate of premature LH surge in the prolonged CC protocol was high (24%). In our study, LH surge occurred in 10% of cases in the CC protocol, coping with incidence obtained by Bhandari et al. who reported LH surge in 9.67% of cases [9]. In our study, the number of oocytes retrieved was significantly higher in the CC group than in the GnRH-ant group; however, the oocyte maturation and fertilization rates were comparable between both groups. In the study of Kang et al. [8], the number of oocytes retrieved was significantly lower in the CC group than in the short protocol group; however, the fertilization and cleavage rates were comparable between both groups. In our study, the number of 8-cell stage embryos was higher in the CC group than in the GnRH-ant group. This could be simply due to higher number of oocytes retrieved in the CC group.
Please cite this article in press as: N.M. Shams-Eldeen et al., Clomiphene citrate in LH surge suppression for women undergoing ICSI, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.01.005
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The pregnancy and implantation rates in our study were comparable between both groups. This agrees with the results of Kang et al. [8] who found comparable outcomes between the CC group and the short protocol group. As the starting gonadotropin dose was similarly high in both protocols, this could be the possible reason behind the slight increase in the incidence of OHSS in the CC group (6%) than in the GnRH-ant group (3.4%). Only 2 cases were cancelled before retrieval (one case in each group), but the other cases were mild and managed symptomatically on an outpatient basis without need of hospital admission. No cases of severe OHSS was reported in the study of Bhandari et al. [9]. This could be attributed to agonist trigger used in their study as well as the fact that the transfer was done in the recipients and not the donors who were stimulated. A strong point of our study lies in being one of the very few studies in which CC was used in a novel protocol for COS in fresh IVF/ICSI cycles (not oocyte donation or frozen/thawed embryos cycles). Matching of both groups for age and BMI represents another strong point. The limitations of our study include lack of randomization and relatively small number of women. 5. Conclusion Adding CC in the mid-to-late follicular phase as an adjuvant to gonadotropins represents a less costly COS which is effective in eliminating the occurrence of premature LH surge without compromising the cycle outcomes in women undergoing ICSI. Conflict of interest There is no conflict of interest. References [1] D.J. McLernon, A. Maheshwari, A.J. Lee, S. Bhattacharya, Cumulative live birth rates after one or more complete cycles of IVF: a population-based study of linked cycle data from 178,898 women, Hum Reprod. 31 (3) (2016) 572–581. [2] K. Stern, Assisted reproductive technology - what’s new and what’s important?, Aust Fam Physician 41 (10) (2012) 762–768.
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Please cite this article in press as: N.M. Shams-Eldeen et al., Clomiphene citrate in LH surge suppression for women undergoing ICSI, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.01.005
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Original Article
Clomiphene citrate in LH surge suppression for women undergoing ICSI Nermeen Mohamad Shams-Eldeen ⇑, Hesham Mahmoud Shalan, Reda Abdel-Hady Hemida, Abdel-Gawad Elmetwally Department of Obstetrics and Gynecology, Mansoura University, Mansoura, Dakahlia, Egypt
a r t i c l e
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Article history: Received 29 December 2017 Revised 9 January 2018 Accepted 12 January 2018 Available online xxxx Keywords: Clomiphene COS LH surge Premature luteinization
a b s t r a c t Objective: To evaluate the effect of adding clomiphene citrate (CC) in the mid-to-late follicular phase as an adjuvant to gonadotropins to suppress luteinizing hormone (LH) surge in women undergoing intracytoplasmic sperm injection (ICSI). Methods: Prospective non-randomized study of 108 women undergoing ICSI and subjected to ovarian stimulation with gonadotropins with addition of CC (50 mg 3 times per day) when a leading follicle reached 14 mm in diameter and continued till the day of human chorionic gonadotropin (HCG) administration. Women subjected to controlled ovarian stimulation (COS) with the gonadotropin-CC protocol (n = 50) were compared with a group of women were to COS with the flexible gonadotropin releasing hormone antagonist (GnRH-ant) protocol (n = 58). Results: Serum LH level on day HCG administration was significantly higher in CC group than in GnRHant group and the incidence of LH surge was higher in CC group than in GnRH-ant group (10% vs 3.4%, respectively) but without statistically significant difference (P = .246). The oocyte maturation and fertilization rates, the biochemical and clinical pregnancy rates, the implantation rate and the ongoing pregnancy rate were comparable in both groups. Conclusion: Adding CC in the mid-to-late follicular phase as an adjuvant to gonadotropins represents a less costly COS which is effective in eliminating the occurrence of premature LH surge without compromising the cycle outcomes in women undergoing ICSI. Ó 2018 Middle East Fertility Society. Production and hosting 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/).
1. Introduction The field of in vitro fertilization (IVF) has grown since 1978 after the birth of Louise Brown and its success has improved over the past 30 years, and about 5 million babies have been born by 2013 worldwide after IVF treatment, and live birth rates per IVF cycle have noticeably increased, particularly for patients younger than 35 years [1]. The need for assisted reproduction techniques (ARTs) is greater than ever, since up to 10% of couples may suffer from some form of infertility and it aims to provide safe and efficient service to increase rate of pregnancy and live birth [2]. The introduction of gonadotropins for ovarian stimulation made a great improvement in IVF success but faced major challenge in
Peer review under responsibility of Middle East Fertility Society. ⇑ Corresponding author at: Department of Obstetrics and Gynecology, Mansoura University Hospitals, Elgomhouria St., Mansoura City 35111, Dakahlia, Egypt. E-mail address: [email protected] (N.M. Shams-Eldeen).
the occurrence of a premature luteinizing hormone (LH) surge and consequent lutinization before ovarian follicle maturation that associates poor oocyte quality, decreased fertilization and implantation rates [3]. Concomitant administration of a gonadotropin releasing hormone (GnRH) agonist or antagonist through controlled ovarian stimulation (COS) protocols, is used to prevent a premature LH surge, and final oocyte maturation and ovulation is typically triggered with a bolus of GnRH agonist, human chorionic gonadotropin (HCG), or both to improve the response, and obtain good quality oocytes and embryos, with maximizing implantation and pregnancy rates [4]. Clomiphene citrate (CC) has been the most widely used for fertility enhancement for 4 decades. It acts as a selective estrogen receptor modulator, as competitive inhibitor of estrogen binding to estrogen receptors. Most evidence suggested primary site of action on hypothalamic estrogen receptors blocking the negative feedback effect of circulating endogenous estrogen resulting in increased plasma level of LH and follicle stimulating hormone (FSH) [5].
https://doi.org/10.1016/j.mefs.2018.01.005 1110-5690/Ó 2018 Middle East Fertility Society. Production and hosting 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/).
Please cite this article in press as: N.M. Shams-Eldeen et al., Clomiphene citrate in LH surge suppression for women undergoing ICSI, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.01.005
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The standard therapy in the past has been the combination of CC treatment in the early follicular phase and subsequent, overlapping gonadotropin stimulation to decrease the amount of required gonadotropins and the costs. In addition, the gonadotropins counteracts the detrimental effects of the CC to the endometrium. One of the major disadvantages is the high occurrences of premature LH surge and consequent lutinization before ovarian follicle maturation and therefore results in the high cancellation rate [6]. Al-Inany et al. [7] showed the beneficial effects of CC during ovarian stimulation with gonadotropins to prevent a premature LH surge in intrauterine insemination (IUI). The rationale is that its antiestrogenic effect may suppress premature LH rise while maintaining a positive influence on ovarian follicle development. In another study, gonadotropins in combination with CC in midto-late follicular phase was compared to short protocol of GnRH agonist-gonadotropins. The gonadotropins-CC protocol resulted in the same pregnancy outcome as short protocol. The novel protocol may take the advantage of eliminating the occurrence of a premature endogenous LH surge [8]. Recently, Bhandari et al. [9] concluded in a pilot study the feasibility of use of continuous CC beyond the usual 5 days with gonadotropins as an effective tool to prevent premature LH surge without compromising oocyte yield. The paucity of studies evaluating efficacy of CC as a tool to prevent premature LH surge during COS pushed us to conduct this study to evaluate the effect of adding CC in the mid-to-late follicular phase as an adjuvant to gonadotropins to suppress LH surge in women undergoing intracytoplasmic sperm injection (ICSI). 2. Method This was a prospective non-randomized study conducted during the period from January 2014 through December 2015 in Mansoura University Fertility Care Unit (FCU), Egypt. The study was approved by the Mansoura Medical Research Ethics committee (code No. MD/28). The participants of this study were chosen from the couples recruited for management of infertility for different causes by ICSI. A written informed consent was taken from each couple selected to participate in the study. The exclusion criteria were: (1) age < 18 or > 38 years; (2) basal serum FSH > 10 IU/L; (3) PCOS; (4) sever endometriosis; or (5) abnormal uterine cavity. 2.1. Controlled ovarian stimulation (COS) protocol In women in the CC group (n = 50), ovarian stimulation using gonadotropin preparation was commenced on day 3 of the stimulation cycle after performing transvaginal sonography (TVS) scan to confirm absence of ovarian cysts. The gonadotropins were given daily by deep intramuscular injection and the starting dose and type depended on the age of the woman, baseline FSH levels, body mass index (BMI) and previous trials. The TVS scan was performed regularly for monitoring follicular development (folliculometry); starting from day 8 of the cycle and repeated every 2–3 days. The dose and type of gonadotropins were modulated according to ovarian response. The CC administration (ClomidÒ, Clomiphene Citrate 50 mg, Marion Merrell, Lebanon; Sanofi-Aventis, Egypt) was started at a dose of 50 mg 3 times per day when a leading follicle reached 14 mm in diameter and continued till the day of HCG administration. Serum LH concentration was measured at day of HCG injection. A control group (n = 58) was selected from women who underwent COS through GnRH antagonist (GnRH-ant) flexible protocol, matched for age and BMI. In these women, ovarian stimulation using gonadotropin preparation was commenced on day 3 of the stimulation cycle after performing TVS scan to confirm absence
of ovarian cysts. The gonadotropins were given daily by deep intramuscular injection and the starting dose and type depended on the age of the woman, baseline FSH levels, BMI and previous trials. TVS scan was performed regularly for monitoring follicular development (folliculometry); starting from day 8 of the cycle and repeated every 2–3 days. The dose and type of gonadotropins were modulated according to ovarian response. The GnRH-ant preparation (CetrotideÒ, Cetrorelix 0.25 mg, Merck Serono, Darmstadt, Germany) was administered subcutaneously in a dose of 0.25 mg/day when a leading follicle reaches 14 mm in diameter and continued till the day of HCG administration. In both groups, if poor ovarian response (< 4 follicles not reaching 18 mm correlated with serum E2 level < 400 pg/ml) was detected after 11–12 days of ovarian stimulation, the couple were informed by the clinicians about the chances of success and the treatment options (to continue treatment, to have intrauterine insemination, whenever indicated, or to be cancelled), and the decision of management depended essentially on the choice of each couple. The cycle was also cancelled when there is a high risk for OHSS. When the leading follicles reach diameter of 18 mm, final oocyte maturation was induced by intramuscular administration of 10,000 IU of HCG. 2.2. IVF/ICSI protocol On the day of HCG injection in both groups, serum LH and serum progesterone levels were assayed as indicators of premature luteinization. After HCG injection by 34–36 h, oocyte retrieval was performed through transvaginal aspiration of follicles under TVS guidance followed by endometrial preparation for embryo transfer (ET). Transfer of embryos was performed transcervically under ultrasound guidance. Luteal phase support was continued by the same regimen which was started on the day of oocytes retrieval until 2 weeks after ET. 2.3. Outcome measures The primary outcome measures of this study were the clinical pregnancy rate and incidence of LH surge. The secondary outcome measures were the ongoing pregnancy rate, implantation rate and Incidence of early OHSS (within 9 days of HCG injection). Clinical pregnancy was defined as presence of at least one intrauterine gestational sac with fetal pole and cardiac activity on TVS scan at 4–6 weeks after embryo transfer (ET). Clinical pregnancy rate was calculated by dividing the number of clinical pregnancies by the number of ET procedures. LH surge was defined as combination of both premature LH rise ( 10 mIU/ml) and premature progesterone rise (>0.8 ng/ml) [10]. Ongoing pregnancy was defined as pregnancy that progressed beyond the critical first trimester (12 weeks gestational age). Implantation rate was calculated by dividing the number of gestational sacs on TVS scan at 4–6 weeks after ET by the number of transferred embryos. Diagnosis and classification of cases of OHSS was reached through clinical picture as well as laboratory results [11]. 2.4. Sample size calculation The sample size was calculated using the computer statistical software G⁄Power 3.1.9.2 (Fisher’s exact test, two-tailed significance, alpha error probability = 0.05, power = 80%, allocation ratio for groups = 1). A sample size of at least 88 women (44 per group) is needed to detect 20% difference between both groups. The estimation of the sample size was based on the previously reported 2% incidence of premature LH surge in flexible GnRH antagonist IVF/ICSI cycle [12], and the reported 22–28% incidence of premature LH surge in clomiphene citrate / gonadotropin IVF/ICSI cycles
Please cite this article in press as: N.M. Shams-Eldeen et al., Clomiphene citrate in LH surge suppression for women undergoing ICSI, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.01.005
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with or without use of GnRH analogues [13,14]. For examination of data from at least 44 patients in each group in the final analysis, it is appropriate to enroll 50 patients in each group. 2.5. Statistical analysis Continuous variables were presented as mean ± standard deviation while categorical variables were presented as frequencies and percentages. The IBMÒ SPSSÒ Statistics, version 20.0 for Windows was used for statistical analysis. P values .05 were considered statistically significant. 3. Results A total of 108 women were included in the study. In 50 women, gonadotropin-CC protocol was used for COS (CC group) and in 58 women, the GnRH-ant flexible protocol was used for COS (GnRHant group). Demographic and hormonal characteristics of both groups are shown in Table 1. The total gonadotropin dose, number of stimulation days and serum E2 level on day HCG administration were comparable in both CC and GnRH-ant groups. Serum LH level on day HCG administration was significantly higher in CC group than in GnRH-ant group. Serum progesterone level on day HCG administration was also higher in CC group than in GnRH-ant group but without statistically significant difference. Endometrial thickness on day HCG administration was significantly lower in CC group than in GnRH-ant group. The incidence of LH surge and the incidence of early OHSS were higher in CC group than in GnRH-ant group but without statistically significant difference (Table 2). The number of cases with cycle cancellation (before oocyte retrieval) and ET cancellation and the causes of these cancellations in both groups are shown in the patient flow (Fig. 1). Although the number of collected oocytes significantly higher in CC group than in GnRH-ant group, oocyte maturation and fertilization rates were comparable in both groups, but the number of 8-cell stage embryos
Table 1 Demographic and hormonal characteristics of the study groups. CC group (n = 50)
GnRH-ant group (n = 58)
P value
Age (years)* BMI (kg/m2)* Duration of infertility (years)*
30.12 ± 5.37 27.49 ± 3.86 5.36 ± 3.08
31.59 ± 4.62 28.09 ± 4.06 6.57 ± 3.97
.104 .378 .135
Type of infertilityy Primary Secondary Previous IVF/ICSIy
33/50 (66.0%) 17/50 (34.0%) 12/50 (24.0%)
39/58 (67.2%) 19/58 (32.8%) 14/58 (24.1%)
.891
Cause of infertilityy Male factor Tubal factor Endometriosis Unexplained Combined Basal serum FSH (mIU/ml)* Basal serum LH (mIU/ml)* Serum prolactin (ng/ml)* Serum TSH (uIU/ml)*
24/50 (48.0%) 19/50 (38.0%) 7/50 (14.0%) 9/50 (18.0%) 9/50 (18.0%) 6.75 ± 1.71 5.51 ± 2.19 12.26 ± 5.44 1.76 ± 0.69
29/58 (50.0%) 27/58 (46.6%) 7/58 (12.1%) 10/58 (17.2%) 11/58 (19.0%) 6.56 ± 1.47 5.40 ± 2.19 11.91 ± 5.14 1.73 ± 0.70
.836 .370 .766 .918 .898 .500 .878 .912 .784
.987
BMI, body mass index. IVF, in vitro fertilization. ICSI, intracytoplasmic sperm injection. FSH, follicle stimulating hormone. TSH, thyroid stimulating hormone. LH, luteinizing hormone. * Expressed as mean ± SD and P value was calculated by the Mann-Whitney U test. y Expressed as frequency and percentage and P value was calculated by the ChiSquare test.
Table 2 COS characteristics of the study groups.
Total FSH dose (IU)* Stimulation days* Serum E2 on day of HCG (pg/ml)* Serum LH on day of HCG (mIU/ml)* Serum progesterone on day of HCG (ng/ml)* Occurrence of LH surgey Endometrial thickness on day of HCG (mm)* Early OHSSy
CC group (n = 50)
GnRH-ant group (n = 58)
P value
2777 ± 578 9.0 ± 1.2 1982 ± 922 3.99 ± 2.44 0.76 ± 0.59
2820 ± 481 8.9 ± 1.1 1903 ± 849 2.30 ± 1.67 0.57 ± 0.39
.807 .703 .675 <.001 .142
5/50 (10.0%) 8.4 ± 1.2
2/58 (3.4%) 9.1 ± 1.0
.246 .002
3/50 (6.0%)
2/58 (3.4%)
.661
COS, controlled ovarian stimulation. E2, estradiol. HCG, human chorionic gonadotropin. FSH, follicle stimulating hormone. LH, luteinizing hormone. * Expressed as mean ± SD and P value was calculated by the Mann-Whitney U test. y Expressed as frequency and percentage and P value was calculated by the Fisher’s exact test.
was significantly higher in CC group than in GnRH-ant group. The number of transferred embryos and the day of ET were comparable in both groups. Also, the biochemical and clinical pregnancy rates, the implantation rate and the ongoing pregnancy rate were similar in both groups (Table 3).
4. Discussion A great improvement in the success of IVF/ICSI was obtained by the use of gonadotropins for ovarian stimulation but this was associated with increased incidence of premature luteinization and poor cycle outcomes owing to premature rise in LH; therefore, the COS protocols were introduced to prevent occurrence premature LH surge and subsequently, improve the cycle outcomes [3]. The CC is considered a first-line therapy for induction of ovulation owing to low cost, oral administration, and minimal adverse effects which represent advantages over injectable medications [15]. So, use of CC is in line with the recent trend toward ‘‘patient-friendly IVF” which is composed of a mixture of four criteria: cost effectiveness, equity of access, minimal risk for mother and child and minimal load for patients [8,16]. The CC used continuously from day 1 or 2 of the menstrual cycle to block estrogen receptors sites at the hypothalamus and pituitary. The CC was used in this way to imitate the action of gonadotropin analogues and prevents an LH surge due to rising estrogen levels, a result of follicular development [9]. CC has a half-life of 24 h or less, and its discontinuation, as in soft stimulation, can cause the initiation of LH surge. This problem can be solved by continuing CC till the day of trigger [17]. Therefore, we conducted this study to evaluate the efficacy of a simplified COS protocol, in which CC was used as an alternative to GnRH antagonist in order to prevent LH surge without affecting the IVF/ICSI cycle outcomes. The CC was administered in the same dose given in the study of Al-Inany et al. [7] due to similar population of their study and our study and also, the accurate dose was not determined from other previous studies. It appears that the antiestrogenic properties of CC did not affect the stimulatory effect of gonadotropins. This was obvious by the fact that the mean E2 levels were even higher in the CC group as was the number of mature follicles in our study group. It was a matter of concern by Singh et al. [17] that the antiestrogenic effect of CC on the genital tract with its prolonged use might alter the
Please cite this article in press as: N.M. Shams-Eldeen et al., Clomiphene citrate in LH surge suppression for women undergoing ICSI, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.01.005
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Fig. 1. Patient flow chart.
Table 3 Outcomes in cases underwent oocyte retrieval and ET in the study groups.
No. of oocytes retrieved* Oocyte maturation rate (%)* Fertilization rate (%)* No. of 8-cell stage embryos* No. 1 2 3 4 5
of transferred embryosy embryo embryos embryos embryos embryos
Day of ETy Day 2 Day 3 Day 4 Day 5 Biochemical pregnancyà Clinical pregnancyà Implantation rate (%)à Ongoing pregnancyà
CC group (n = 44)
GnRH-ant group (n = 53)
P value
11.1 ± 6.6 83.5 ± 15.0 70.6 ± 19.8 6.2 ± 3.9
8.4 ± 5.2 80.5 ± 22.0 72.5 ± 24.1 4.3 ± 3.0
.033 .953 .444 .009
3/44 (6.8%) 6/44 (13.6%) 23/44 (52.3%) 10/44 (22.7%) 2/44 (4.5%)
9/53 (17.0%) 6/53 (11.3%) 24/53 (45.3%) 10/53 (18.9%) 4/53 (7.5%)
.215 .765 .544 .802 .686
2/44 (4.5%) 28/44 (63.6%) 12/44 (27.3%) 2/44 (4.5%) 16/44 (36.4%) 14/44 (31.8%) 22/134 (16.4%) 12/14 (85.7%)
3/53 (5.7%) 41/53 (77.4%) 7/53 (13.2%) 2/53 (3.8%) 19/53 (35.8%) 16/53 (30.2%) 27/153 (17.6%) 14/16 (87.5%)
1.000 .178 .122 1.000 .958 .863 .782 .886
ET, embryo transfer. OHSS, ovarian hyperstimulation syndrome. * Expressed as mean ± SD and P value was calculated by the Mann-Whitney U test. y Expressed as frequency and percentage and P value was calculated by the Fisher’s exact test. à Expressed as frequency and percentage and P value was calculated by the Chi-Square test.
endometrial receptivity as their protocol used in oocyte donors and when sequential transfer is planned. Although it was statistically significant, there was slight difference (0.6 mm) in the mean endometrial thickness between the CC group and GnRH-ant group in our study. However, the endometrial thickness obtained in our study was higher than that was obtained by Al-Inany et al. [7]. This difference may be attributed to higher doses of gonadotropins which counteract the deleterious effect of CC on the endometrium. On the other hand, the mean endometrial thickness in our study was lower than that was obtained by Kang et al. [8] (nearly 11 mm in both groups). The mean serum LH level on the HCG injection day was significantly higher with the CC protocol in our study, but not to the
extent of occurrence of LH surge (3.99 mIU/ml). This level is lower than that was obtained by Bhandari et al. [9] (6.68 mIU/ml), but higher than that was obtained by Kang et al. [8] (2.51 mIU/ml). These contradictory results could be explained by the lower CC dose (50 gm/d) used by Bhandari et al. [9], and the lower total gonadotropin dose (2033 ± 555 IU) given in the study of Kang et al. [8]. Also, in contrast to our study, Kang et al. al [8] had found significant lower LH level in the CC protocol group than in the short protocol group (2.51 vs 3.64 mIU/ml). This could be explained by the fact that short GnRHa protocol has initial flare up effect but the GnRH antagonist and CC-gonadotropin protocols do not have this effect. Singh et al. [17] have used CC in their study from the beginning of stimulation till the day of trigger in oocyte donors and compared it with the other donors, receiving GnRH-ant flexible protocol. Following the same idea, but we started before publishing of the Singh et al. [17] results, seemed good to allow us for comparing new protocol with the old established one using GnRH-ant in flexible manner (when follicle reaches >14 mm), and from the physiological point that CC when given with high serum E2 will benefit its effect in prevention of LH surge, that was a novel trend and need to be investigated further. The mechanism of triggering LH surge in the hypothalamus is not very clear and it has been reported that LH surge occurs in nearly 20–25% of CC and/or gonadotropin cycles [18]. The extended use of CC in a dose of 100 mg for 8 days (from cycle day 3 to 10) was by applied by Matsaseng et al. [19] to prevent LH surge and thus avoid the use of GnRH analogues and reduce the cost; however, the rate of premature LH surge in the prolonged CC protocol was high (24%). In our study, LH surge occurred in 10% of cases in the CC protocol, coping with incidence obtained by Bhandari et al. who reported LH surge in 9.67% of cases [9]. In our study, the number of oocytes retrieved was significantly higher in the CC group than in the GnRH-ant group; however, the oocyte maturation and fertilization rates were comparable between both groups. In the study of Kang et al. [8], the number of oocytes retrieved was significantly lower in the CC group than in the short protocol group; however, the fertilization and cleavage rates were comparable between both groups. In our study, the number of 8-cell stage embryos was higher in the CC group than in the GnRH-ant group. This could be simply due to higher number of oocytes retrieved in the CC group.
Please cite this article in press as: N.M. Shams-Eldeen et al., Clomiphene citrate in LH surge suppression for women undergoing ICSI, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.01.005
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The pregnancy and implantation rates in our study were comparable between both groups. This agrees with the results of Kang et al. [8] who found comparable outcomes between the CC group and the short protocol group. As the starting gonadotropin dose was similarly high in both protocols, this could be the possible reason behind the slight increase in the incidence of OHSS in the CC group (6%) than in the GnRH-ant group (3.4%). Only 2 cases were cancelled before retrieval (one case in each group), but the other cases were mild and managed symptomatically on an outpatient basis without need of hospital admission. No cases of severe OHSS was reported in the study of Bhandari et al. [9]. This could be attributed to agonist trigger used in their study as well as the fact that the transfer was done in the recipients and not the donors who were stimulated. A strong point of our study lies in being one of the very few studies in which CC was used in a novel protocol for COS in fresh IVF/ICSI cycles (not oocyte donation or frozen/thawed embryos cycles). Matching of both groups for age and BMI represents another strong point. The limitations of our study include lack of randomization and relatively small number of women. 5. Conclusion Adding CC in the mid-to-late follicular phase as an adjuvant to gonadotropins represents a less costly COS which is effective in eliminating the occurrence of premature LH surge without compromising the cycle outcomes in women undergoing ICSI. Conflict of interest There is no conflict of interest. References [1] D.J. McLernon, A. Maheshwari, A.J. Lee, S. Bhattacharya, Cumulative live birth rates after one or more complete cycles of IVF: a population-based study of linked cycle data from 178,898 women, Hum Reprod. 31 (3) (2016) 572–581. [2] K. Stern, Assisted reproductive technology - what’s new and what’s important?, Aust Fam Physician 41 (10) (2012) 762–768.
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Please cite this article in press as: N.M. Shams-Eldeen et al., Clomiphene citrate in LH surge suppression for women undergoing ICSI, Middle East Fertil Soc J (2018), https://doi.org/10.1016/j.mefs.2018.01.005