The effects of low-dose human chorionic gonadotropin for luteal phase support on pregnancy outcomes in poor ovarian responders: a randomized clinical trial

Purpose: The question that remains is, does changing the type of luteal phase support (LPS) improve the pregnancy outcomes in patients with poor ovarian response (POR) diagnosis?. Therefore, this study was designed to investigate and compare the eciency of different methods of luteal phase support (progesterone alone or hCG alone and the combination of progesterone with hCG) in these patients. Methods: This randomized clinical trial evaluated three hundred seventy ve patients who were diagnosed as POR on the basis of Bologna criteria undergoing intracytoplasmic sperm injection- embryo transfer (ICSI-ET) cycles at Royan institute from November 2015 to June 2019. The patients were allocated randomly into three different LPS groups on the day of oocyte pickup. In rst group, 1500 IU of hCG IM on the ET day, as well as 4 days after that were administrated. In the second group, the patients received 1500 IU of hCG IM on the ET day, as well as 3 and 6 days after the ET along with vaginal suppositories 400 mg twice daily. For the third group, only vaginal suppositories twice daily was administrated from the day of oocyte pick up until the pregnancy test day.The clinical pregnancy, miscarriage and live birth rates were the main outcomes. Results: The data analysis showed that the three groups were comparable. In the following, there is no signicant difference in terms of implantation, clinical pregnancy, and miscarriage and live birth rates among groups. The twin pregnancy rate in the hCG-only group was higher than those of in the other two groups, although this difference was not statistically signicant (P=0.06). Conclusion: The type of LPS does not improve the pregnancy and live birth rates in POR patients. A multi-center clinical trial is warranted to conrm or refute these ndings. Trial registration: The study was registered in the clinicaltrial.gov site on 14 June 2015. (NCT02798653 at www. clinicaltrials.gov, registered prospectively).


Introduction
The requirement of luteal phase support (LPS) in assisted reproduction cycles is well established, but the issues that remain controversial are the ideal drug, the appropriate route of administration, the timing and duration of support [1]. The progesterone production in luteal phase is a key component that is necessary for the successful implantation of the developing embryo. Different route of progesterone administration was used including oral, intramuscular, subcutaneous and vaginal. Recently, different types of vaginal progesterone (suppositories, gels and tablets) are common choice for LPS; however, those may be associated with different side effects and discomfort of patients [2]. The need for simpli ed treatment approaches which will lessen the treatment burden of IVF is self-evident; therefore, clinical physicians in ART medicine usually recommend drug therapy with lower doses and repeated use. For example, the use of only two dose of human chorionic gonadotropin hormone (hCG) injection for luteal phase support is more comfortable and tolerable than administration of progesterone daily, either vaginally or by injection for patients.
A number of clinical trials have been compared the effect of hCG and progesterone for luteal phase support; the results of a recent meta-analysis showed that there were no statistically signi cant difference among different luteal phase support methods in terms of clinical pregnancy, miscarriage and ongoing pregnancy rates; therefore, it seems that progesterone alone is the best strategy due to lower risk of OHSS [3]. Recently, use of micro-dose of hCG without exogenous progesterone for luteal phase support has been considered and it is hypothesized that corpus luteum produces other hormones than estrogen and progesterone, which are essential for endometrial preparation and optimization of the environment for embryo implantation and development [4].
Due to the fact that in patients with poor ovarian response (POR) diagnosis, there is no risk of OHSS and on the basis of the novel concept that suggested two boluses of 1500 IU hCG with no additional luteal support revert the luteolysis after a GnRHa trigger in the normo-responder patient [5], designing a clinical trial study to evaluate this simple and patients friendly method for luteal phase support in POR patients is gaining interest. Therefore, this clinical trial was designed to investigate and compare the e ciency of different methods of luteal phase support (progesterone alone or hCG alone and the combination of progesterone with hCG) in these patients. It is hoped that the results of this study would be useful in improving the clinical pregnancy rate and in making decision for the best method of luteal phase support in these women.

Methods
This randomized clinical trial was carried out to investigate the e cacy of different luteal phase support methods in patients with poor ovarian response (POR) undergoing intracytoplasmic sperm injection (ICSI) cycles in Royan institute from November 2015 to June 2019. The study protocol is approved by the Institutional Review Board and Ethics Committee of Royan institute. The study is conducted according to the Declaration of Helsinki for medical research. All participants provided informed consent after receiving explaining the purpose of the study. The trial protocol was registered prospectively in Clinicaltrials.gov site (NCT02798653).
All the patients who diagnose as poor ovarian responders (POR) based on the Bologna criteria were eligible for participation in this study. In order to de ne the poor response in IVF, at least two of the following three features must be present: (i) advanced maternal age (over 40 years ); (ii) a previous POR (total retrieved oocytes less than three oocytes using conventional protocols) and (iii) an abnormal ovarian reserve test (ORT) (antral follicle count of less than 5 on menstrual cycle day 2-3; and basal serum follicle stimulating hormone (FSH) concentration between 10 and 19 IU/l or serum anti-Müllerian hormone level less than 1 ng/ml). Two episodes of POR after maximal stimulation are su cient to de ne a patient as poor responder in the absence of advanced maternal age or abnormal ORT. The advanced maternal age over 45 years old, cigarette, alcohol and drug addiction, diagnosis of ovarian failure including basal FSH above 20 IU/l or no antral follicle by ultrasound examination, severe male factor (azoospermia) diagnosis, severe endometriosis and the presence of hydrosalpinges, uterine factor (polyps, myoma and previous myomectomy,…), the patients with cardiovascular disease and/or uncontrolled systemic or endocrine diseases and repeated implantation failures and repeated miscarriages cases were excluded from study.
The ovarian stimulation is performed with GnRH agonist stop or antagonist protocols for the eligible patients as explained in details previously. In both protocols, controlled ovarian stimulation is started on day 2 of menstrual cycle with 225 IU recombinant FSH (Gonal-F ® ; Serono Laboratories Ltd., Geneva, Switzerland) and 75 IU hMG (Menopur ® ; Ferring). The doses of gonadotropins were adjusted as ovarian response in the ultrasound monitoring and nal oocyte triggering will be done with 10000 IU of hCG (Choriomon®; IBSA). If there were or more dominant follicles, oocytes retrieval will be done under transvaginal ultrasound guidance 32-34 hours after hCG administration.
On the day of oocyte pickup, patients are allocated randomly (by the blocked randomization method) into three groups to receive three different luteal support protocols. Permuted block randomization was prepared by the methodological advisor according to a computer-generated list. The patients' enrolment and assignment to different groups were carried out by a researcher midwife in the clinic. Each patient was participated in the study only once and if she had written consent. The researcher who followed the results of patients' treatment and the researcher who analyzed the data were kept masked to the type of LPS regimen.
In rst group, 1500 IU of hCG IM on the embryos transfer (ET) day, as well as 4 days after that were administrated for luteal phase support. In the second group, the patients received 1500 IU of hCG IM on the ET day, as well as 3 and 6 days after the ET along with vaginal suppositories (Cyclogest ® , Actavis, UK) 400 mg twice daily. For the third group, only vaginal suppositories (Cyclogest ® , Actavis, UK) 400 mg twice daily was given for luteal support from the day of oocyte pick up until the pregnancy test day. Intracytoplasmic sperm injection was performed for all metaphase II oocytes. Embryos were cultured in a commercially available culture medium until the day of transfer. The obtained embryos at cleavage stage were replaced by an embryo transfer catheter (Guardia™, Access ET Catheter, Cook Medical), 2 or 3 days after oocytes retrieval. The serum ß-hCG level was checked 2 weeks after ET to con rm positive pregnancy test. The vaginal progesterone was administrated in all patients who become pregnant in all three groups until the 10th week of pregnancy. The pregnancy outcomes are compared among three study groups.

Outcome measures
The primary outcome was implantation (the number of embryos transferred for each patient), chemical pregnancy (only positive ß-hCG test) and clinical pregnancy (the presence of a gestational sac with fetal heart beat on vaginal ultrasound) rates. The secondary outcomes included early miscarriage (the spontaneous loss of a clinical pregnancy under 12 weeks of gestation) and late miscarriage (the spontaneous loss of a clinical pregnancy between 12 and 20 weeks of gestation) and live birth (the delivery of a living child irrespective of the duration of pregnancy) rates.

Statistical Analysis
Sample size was calculated on the basis of previous related studies by using the Power Analysis and Sample Size (PASS) software Version 11. A sample size of 125 patients was required in each group at a signi cance level of 0.05 and a power of 80%. Statistical analysis was done using the Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, USA) version 23.0. The continuous variables were compared among groups by the one-way analysis of variance (ANOVA) and were presented as mean ± standard deviation (SD). The chi-square test was applied for comparing the categorical variables among groups and the results were reported as numbers/percentages. The statistical signi cant level was considered at p value <0.05.

Results
A total of 524 women were evaluated to participate in the study. 38 patients did not consent to participate and 111 patients did not reach to ET stage; nally, 375 patients allocated into three groups randomly. 125 patients were received only two boluses of 1500 IU hCG (intervention group1), 125 patients were received three boluses of I500 IU hCG plus vaginal progesterone daily (intervention group 2), and 125 patients were received only vaginal progesterone daily (control group) (Fig. 1).
The analysis of data showed that the three groups were comparable and there was no signi cant difference in terms of age, BMI, duration of infertility, and other basic characteristics of patients among three groups ( Table 1).
The ovarian stimulation outcomes are presented in Table 2. No difference existed among the three groups with respect to the type of COH protocol, total dose of gonadotropins and the duration of the ovarian stimulation, the number of retrieved oocytes, fertilization rate, number of good quality embryos transferred, and endometrial thickness on the day of ovum pickup. Coincidentally the ratio of 2 and 3-day cleavage stage embryos in the hCG-only group had a statistically signi cant difference in compared to other groups (P =0.007). Table 3 shows the pregnancy outcomes in three groups. The analysis demonstrated that the implantation, clinical pregnancy, miscarriage and live birth rates were similar between groups.
The twin pregnancy rate in the hCG-only group was higher than those of in the other two groups, although this difference was not statistically signi cant (P=0.06).

Discussion
The present study was conducted to response the question of whether changing the type of luteal phase support (LPS) in patients with POR diagnosis improves the rates of clinical pregnancy and live birth. The results of the study showed that the type of luteal phase support did not affect the clinical pregnancy and live birth rates in POR patients, just the rate of twin pregnancy in the hCG -alone group was a little more than progesterone-alone group.
Fundamentally, LPS has been demonstrated as a vital part in IVF cycles because ovarian stimulation disrupts the particular function of corpus luteum (CL) by different mechanisms. The formation of several mature ovarian follicles, E 2 levels reach to supraphysiological ranges speci cally during the follicular phase; moreover, progesterone levels increase in early days of luteal phase due to numerous CLs affected by triggering doses of hCG. Thus LH secretion decompressed during luteal phase afterwards, CL support by pituitary LH will not precede [6,7]. Notwithstanding the foregoing, it is important to realize that the CL does not require supraphysiologic levels of LH/hCG to secrete high values of progesterone [8]. During the natural menstrual cycle, the LH level in the luteal phase rarely rises over 5-10 IU/L and most commonly is capable to elicit P levels up to 25-35 nmol/L. When ten CLs are exist, each will secrete P in amounts similar to the natural menstrual cycle when exposed to physiologic concentrations of LH/hCG [8].
Collectively, this results in high concentration of P. However, this condition occurs when the ovarian response is normal or excessive, and therefore in POR patients due to limited AFC and growing follicles and the lack of existence of suitable CL, the possibility of hormonal defects in the LPS is higher.
Several protocols have been developed to correct this hormonal inadequacy in IVF cycles, based on the substances used or the route of administration. The development of new regimens for LPS based on ''low-dose'' or ''microdose'' hCG supplementation was considered recently. In preliminary studies, four injections of 1500 to 2500 IU of hCG were administrated after nal oocyte triggering with 10000 IU of hCG [9]; since then, several studies have examined different methods of luteal phase support using hCG alone or with progesterone in normal patients or donor patients [10][11][12][13]. With these regimes, the LH/ hCG concentration in the luteal phase often reaches more than ve to ten times that observed in the normal menstrual cycle which make powerful luteal phase support; however, signi cantly increase the risk of OHSS [4]. In a recent Cochrane review study reported that the administration of hCG alone or in combination with progesterone to support the luteal phase should be avoided due to increase the risk of OHSS [3].
Moreover, a number of studies have suggested that in cases where GnRH agonist is used for the nal oocyte triggering, very low doses of hCG of 100 to 150 IU per day could be appropriate option for the LPS [14,15]. Progesterone levels in the middle of the luteal phase in this procedure were similar to the use of progesterone with 6500 IU of hCG to induce ovulation [8]. The limitation of this strategy that prevents from being applied to routine procedures is that the di culty HCG dilution over time with normal saline to achieve a dose of 100 to 150 units and daily injection for the patient [4].
Considering that there is no risk of OHSS in POR patients, it seems that the study of this new LPS regime with low-dose hCG for improving pregnancy rate is valuable. Furthermore, women appear to prefer lowdose hCG administration to exogenous P administration, especially by the vaginal route.
In present study, when twin pregnancy rate was compared among groups, it was higher in the hCG-only group than that of in the P-only and hCG + P groups; however, it was close to signi cant level. In this regard, Var et al. reported that multiple pregnancies rate were signi cantly higher in the hCG+ P group than in the P-only and E 2 + P groups [11]. Similar to their ndings, Ludwig et al. also found that multiple pregnancy rate were higher in the hCG +P group than in the P-only group [10]. However, Ghanem and colleagues reported no statistical difference among the groups in terms of multiple pregnancies; they determined a higher tendency in the E 2 + P and hCG + P groups compared with the P-only group [16]. It is likely that the use of hCG for LPS are involved in increasing the rate of multiple pregnancies and more studies are necessary in this regard.
The main limitation of the study is that we did not consider the good quality euploid blastocysts and unique COH protocol as inclusion criteria. Although, in data analysis the number and quality of transferred embryos and the proportions of two COH protocols were similar among groups, it is recommended that these points be considered in future studies to eliminate these confounding factors.
In conclusion the present study is the rst clinical trial to compare the effect of low-dose hCG with routine protocol for LPS in patients with POR diagnosis on the basis of the Bologna criteria. We found the same pregnancy and live birth rates in different regimes for LPS. Interestingly, the use of two bolus of low-dose hCG 1500 was associated a slight increase in multiple pregnancies. We recommend a multi-center clinical trial with a larger sample size in this eld to con rm or refute our ndings. The eligible patients signed written informed consent prior to participation in the study.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request.
Authors' contributions TM and FR designed the research. AA, FR and SHKH contributed in patient's selection, data collection, interpretation of data and manuscript writing/editing. AA and TM wrote the manuscript. ZZ helped in the analysis of the data. All authors read and approved the nal manuscript.   The study owchart