Introduction
Numerous modifications and advancements have been made to in vitro fertilization (IVF) since its inception, relating to aspects such as ovarian stimulation and catheter selection. Despite these improvements, the implantation rate (IR) and clinical pregnancy rate (CPR) among women undergoing embryo transfer (ET) have consistently failed to meet expected outcomes. This discrepancy is attributed to various factors, including endometrial receptivity and embryo quality. Extensive research has been conducted to understand and improve the effectiveness of ET following IVF [1].
For successful embryo implantation in humans, the endometrium must demonstrate functional competency and receptivity [2]. Challenges arise from both endometrial and embryonic factors, which can obstruct the implantation process that is essential for pregnancy. Implantation failure can result from either a poor-quality embryo or issues with endometrial receptivity [3]. Factors such as endometrial thickness, uterine artery blood flow, and endometrial perfusion all contribute to endometrial receptivity and can potentially reduce the IR in IVF/intracytoplasmic sperm injection (ICSI) cycles [4].
Elevated impedance in uterine blood flow can lead to inadequate endometrial development and a thinning of the uterine lining. Optimal blood flow to the uterus is crucial for the healthy growth and receptivity of the endometrium [4]. Recent research has indicated that a high uterine artery pulsatility index (UtA-PI) is associated with a reduction in this flow, potentially resulting in implantation failure and pregnancy complications [5]. Women undergoing IVF/ICSI cycles with an elevated UtA-PI face relatively low rates of implantation and clinical pregnancy. Pharmacological intervention to address this issue could improve the outcomes of IVF/ICSI cycles [6].
Calcium channel blockers and vasodilators can increase endometrial thickness, promote uterine relaxation, and improve endometrial receptivity, thus potentially increasing the likelihood of a successful assisted pregnancy [7]. A range of vasodilators, including nifedipine, isosorbide mononitrate, pentoxifylline, glyceryl trinitrate, sildenafil, and amlodipine, have been utilized to address issues with endometrial thickening and uterine relaxation in the context of assisted reproductive technology (ART) [7].
Amlodipine is a long-acting oral dihydropyridine calcium channel blocker that is notable for its low incidence of adverse drug reactions associated with vasodilation [8]. It reduces the initial influx of calcium by blocking voltage-dependent L-type calcium channels [8]. As intracellular calcium decreases, the contractility of vascular smooth muscle is reduced, while smooth muscle relaxation and vasodilation are increased. Additionally, amlodipine improves endothelial function in blood vessels of individuals with hypertension, improving blood flow and contributing to its therapeutic efficacy [9]. The purpose of this study is to examine the effects of amlodipine administration prior to ET on uterine PI, resistance index (RI), and ET outcomes.
Methods
1. Study design, setting, and population
The study was conducted as a randomized clinical trial at our fertility center, adhering strictly to the principles of the Declaration of Helsinki and the Consolidated Standards for Reporting Trials (CONSORT) statement. Participants were recruited based on predefined inclusion and exclusion criteria. All prior unsuccessful cycles, including frozen embryo transfer (FET) cycles, were considered, and the women received a uniform hormone treatment protocol. After providing informed consent, participants were formally enrolled in the study. The Research Ethics Committees of Tabriz University of Medical Sciences conducted a thorough review and granted approval for the study, documented under the reference number IR.TBZMED.REC.1401.965.
2. Allocation and intervention
The women were randomly assigned to receive either amlodipine or placebo in a 1:1 ratio, based on a computer-generated randomization list. Allocation numbers were enclosed in sealed envelopes, which a non-participating nurse opened. Women in the amlodipine group were administered 5 mg of oral amlodipine (Zahravi Pharmaceutical Co.), while those in the placebo group received microcrystalline cellulose over the same period. A nurse not involved in the study managed the medication administration for both groups. To maintain blinding, the participants, healthcare providers, and laboratory personnel remained unaware of the group assignments. The codes for the treatment groups were revealed to the investigators only after the study and statistical analysis were completed.
3. Sample size determination
A study by Sohrabvand et al. [10] reported an overall pregnancy rate of 13.6% in the control group compared to 36% in the intervention group. We calculated a total sample size of 159 women, considering a type I error of 0.05, a power of 0.8, and an expected attrition rate of 20% for each group.
4. Clinical trial registration
This clinical trial was registered with the Iranian Registry of Clinical Trials (IRCT), with the assigned registration number IRCT20101227005485N11, on February 6, 2023. The trial protocol has been published at https://en.irct.ir/trial/68330.
5. Eligibility criteria
For inclusion, participants were required to be aged 20 to 45 years (inclusive); have a body mass index (BMI) of 25 to 30 kg/m2; be scheduled for cleavage-stage FET, with vitrification occurring on day 3, during a hormone replacement therapy (HRT) cycle; have at least one uterine PI exceeding 3, of the two measurements taken on the initial day of monitoring; and have a history of at least one failed ET. Furthermore, participants had to be candidates for a simple transfer test, involving the passage of a flexible or rigid catheter through the cervix without the need for anesthesia or Pozzi forceps, and to provide free written informed consent.
The exclusion criteria were as follows: active smokers (defined as at least one cigarette per day at the time of study registration); oocyte recipient patient; those scheduled to undergo pre-implantation genetic screening; patients with stage 3 or 4 endometriosis, or with severe adenomyosis or submucous myoma; individuals with contraindications to amlodipine use (severe angina pectoris, cyclosporine treatment, myocardial infarction less than 1 month prior, severe hepatic insufficiency, hypotension, or hypersensitivity to amlodipine or dihydropyridine derivatives); those concurrently using cytochrome P450 family 3 subfamily A member 4 (CYP3A4) enzyme inhibitors or inducers; those being treated for hypertension during the initial monitoring period; and participants in other human intervention research.
6. Procedures
Demographic data, including age, BMI, and duration of infertility, were extracted from the records of each participant, and a data collection form was completed for every individual. All participants received the standard FET protocol. To assess PI and RI, each woman underwent transvaginal color Doppler examination of the right and left uterine arteries on day 1 or 2 of her menstrual cycle. A single sonographer conducted all transvaginal color Doppler examinations.
7. Drug intervention
Following the cessation of ovarian hormone production, HRT was initiated with daily administration of 6 mg of estradiol. This regimen continued until a pregnancy test was performed. If the result was positive, HRT was maintained for the first 12 weeks of amenorrhea at the same dosage before being gradually tapered. In the event of a negative pregnancy test, HRT was slowly discontinued. The endometrial pattern and vascular perfusion were closely observed between the first and second days of the menstrual cycle. One group was administered 5 mg of oral amlodipine (Zahravi Pharmaceutical Co.), while the other received a placebo consisting of microcrystalline cellulose. The assigned treatment was maintained until pregnancy testing was conducted. If the result was positive, treatment (amlodipine or placebo) was continued for a total of 7 weeks.
8. Monitoring and evaluation
To establish baseline values for each patient, PI and RI were measured prior to the administration of amlodipine. These indices were measured again immediately before FET to assess the impact of amlodipine. Pulsed-wave Doppler ultrasound was performed on the first or second day of the participant’s menstrual cycle. Amlodipine treatment was initiated for the medication group. The evaluation of RI and PI in the uterine artery was conducted at the level of the cervical opening of the cervix into the body of the uterus, with concurrent visualization of the uterus and cervix. Doppler ultrasound was employed to assess endometrial blood flow, focusing on four areas: zone 1, where blood vessels enter the endometrial/myometrial tissue surrounding the endometrium; zone 2, where vessels enter the hyperechoic endometrial margin; zone 3, where vessels enter the internal hypoechoic zone of the endometrium; and zone 4, where vessels enter the endometrial cavity (Figure 1). Doppler sonography was performed using a vaginal probe operating at 7.5 MHz on an Affiniti 70 ultrasound machine (Philips), with the uterus in a sagittal view.
9. Embryo transfer
FET was performed 4 days after the initiation of progesterone therapy. The Kitazato kit was used to thaw the embryos and reassess their quality. The morphological criteria for embryo scoring at both cleavage and blastocyst stages were based on the Istanbul consensus [11] and the Gardner scoring criteria [12]. In accordance with the former, all grade 1 cleavage-stage embryos were defined as being of good quality. An embryo was considered to have survived if at least 50% of its cells were intact; those with a minimum of six cells and less than 20% intracellular fragmentation were also considered good quality. After thawing, the embryos deemed suitable for use were cultured for 2 hours. Subsequently, they were transferred into the uterine cavity under ultrasonographic guidance, with each procedure involving the transfer of 2–3 embryos. The transfers were conducted smoothly using a Cook catheter and ultrasound guidance, without the need for a tenaculum. Amlodipine administration was continued until pregnancy testing produced a positive result. Ultimately, we compared the mean values for blood flow indices in the uterine artery, the spiral artery supply zone, the endometrial thickness, and the clinical and chemical pregnancy rates between groups.
10. Outcomes
In this study, the primary outcomes were two indices of uterine artery blood flow: PI and RI. To measure these parameters, the Doppler wave was activated, and the angle of incidence was adjusted to record two comparable flow waves in the uterine artery. Simple semiquantitative methods were used to analyze the Doppler waveforms, incorporating the measurement of spectral characteristics such as the PI and RI. Radiologists utilized Doppler ultrasound to measure these characteristics for each participant in both study groups. Secondary outcomes included IR, as well as clinical and chemical pregnancy rates. IR was determined by dividing the number of gestational sacs by the number of embryos transferred to the uterus. Fourteen days after transfer, beta-human chorionic gonadotropin (β-hCG) testing was performed to detect chemical pregnancy. Clinical pregnancy was confirmed if ultrasound revealed a gestational sac 4 weeks after the transfer.
11. Statistical analysis
SPSS ver. 16 (SPSS Inc.) was utilized for data analysis. The chi-square test, the independent t-test, and analysis of variance were used to examine differences in variables between the two groups. A p-value of less than 0.05 was considered to indicate statistical significance.
12. Ethical approval
The Ethics Committee has no objections to the implementation of the study project. All methods were conducted in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects or their legal guardians under the Ethical Approval and Consent to Participate section.
Results
Between February and November 2023, 159 women were screened. Of these, 20 did not meet the selection criteria, and 25 declined to participate. Consequently, 114 participants were randomly assigned to two groups. Seven patients from the amlodipine group and seven from the control group were later excluded due to unwillingness to continue. Ultimately, 100 cases were analyzed, with 50 in the treatment group and 50 in the placebo group. The study flowchart is presented in Figure 2. No significant differences were found between groups regarding demographic variables such as the duration of infertility, BMI, age, endometrial thickness, the number of embryos transferred, and the frequency of fertility treatments (Table 1). This indicates that the randomization of the study groups was successful. The dependent variables exhibited normal distributions. Notably, 18% of placebo recipients tested positive for β-hCG, compared to 26% of medication recipients; nevertheless, this difference was not statistically significant (p=0.472). Additionally, 12% of the placebo group exhibited a gestational sac, while 22% of the medication group did so; this difference was also statistically insignificant (p=0.287). However, a significant distinction was observed between groups regarding the ultrasound pattern of the endometrium (p=0.007). A significant difference was also noted in the distribution of endometrial blood supply zones between the drug and placebo recipients (p<0.001) (Table 2). The UtA-PI and the uterine endometrial RI were assessed in both groups. The UtA-PI was 2.54±0.52 in the drug group and 3.5±0.89 in the placebo group, representing a significant difference (p<0.001). The endometrial RI was 0.83±0.05 in the treatment group and 0.91±0.053 in the placebo group, which was also a statistically significant finding (p<0.001) (Table 3, Figure 3). Pregnancy outcomes are presented in Table 4.
Discussion
The success of ART cycles is highly dependent on the receptivity of the endometrium [13]. The objective of endometrial preparation is to establish optimal conditions for embryo implantation. With advancements in ART, increasing attention has been dedicated to identifying the ideal state of endometrial preparation to maximize the chances of successful pregnancy [14].
The spiral arteries originate from the uterine arteries, and the resistance levels of these vessels are directly related. Consequently, assessing the resistance in the uterine arteries can provide valuable insights into the potential for endometrial implantation. Several studies have indicated that when the PI of the uterine arteries exceeds 3.2, IRs are notably low [15-17]. This suggests that procedures such as ET or intrauterine insemination should be deferred. Blood flow in the uterine arteries is crucial for promoting endometrial growth, supporting the luteal phase, and influencing the duration of pregnancy. Previous research has established a strong relationship between Doppler measurements of the uterine arteries and various complications, including recurrent spontaneous abortion, repeated implantation failure, preeclampsia, and intrauterine fetal growth restriction [18-20]. Timely intervention based on these measurements can help reduce pregnancy complications and improve outcomes for both mother and fetus. On Doppler ultrasound, a high PI is an independent risk factor for implantation failure and pregnancy complications [13]. Thus, it is worthwhile to investigate pharmacological interventions to reduce the UtA-PI prior to ET. These interventions could include various drug classes, such as calcium channel blockers and vasodilators [7,21].
The recommended maximum daily dosage of amlodipine is 10 mg [22]. Amlodipine is characterized by high bioavailability, minimal fluctuations in plasma concentration, and the achievement of a stable plasma concentration within 7 to 8 days following the initiation of a 5 mg daily oral dose. Research indicates that amlodipine exposure during pregnancy does not increase the incidence of embryonic or fetal abnormalities relative to other antihypertensive medications or untreated maternal hypertension [23-25]. A recent meta-analysis also supports the safety and efficacy of amlodipine for managing hypertension during pregnancy [26]. Amlodipine effectively lowers blood pressure and improves vascular function by inducing smooth muscle relaxation and dilating blood vessels. Given its efficacy in reducing blood pressure and supporting vascular health, amlodipine is recommended as an appropriate antihypertensive agent for individuals with moderate to severe hypertension [27].
This study is the first to evaluate the effects of amlodipine on uterine PI, RI, and successful ET in individuals with previous implantation failure. The findings indicate that amlodipine reduced blood flow indices in the uterine artery, although no significant differences were observed in chemical or CPRs between the treatment and control groups. As this is the first study to explore the potential benefits of amlodipine in FET, its findings cannot be compared with previous research. While several studies have investigated the use of additional pharmacological agents to improve ET outcomes, the results have been inconclusive [28]. Two adrenoceptor agonists, terbutaline and ritodrine, are recognized for their capacity to relax the uterus by reducing smooth muscle tension. However, a large randomized trial found no significant differences in IR or CPR between the intervention and control groups [29,30].
Limited research has compared FET and fresh blastocyst transfer regarding UtA-PI in IVF recipients [31-33]. Two studies suggested that patients undergoing frozen blastocyst transfer exhibit superior uterine perfusion and fetal growth relative to those experiencing fresh blastocyst transfer [34,35]. In a study by Choux et al. [33], the PI was significantly higher in fresh ET recipients compared to those who conceived naturally (p=0.001) and significantly lower in the FET group compared to the fresh ET group (p=0.001) [31]. Additionally, two other studies reported lower UtA-PI values in FET as opposed to fresh blastocyst transfer recipients, corroborating these findings [34,36].
Several methods can improve the transfer quality during the final phase of ART. These methods include soft ET, ultrasound guidance, and the use of specific pharmaceutical agents [36]. Various pharmacological strategies are available to improve IVF outcomes, such as the administration of growth hormone-releasing factor or growth hormone, letrozole, transdermal testosterone, oral L-arginine, and nifedipine [29], as well as the application of platelet-rich plasma [37] and granulocyte-macrophage colony-stimulating factor [38]. A wide range of factors associated with different aspects of assisted reproduction can influence the pregnancy rate in IVF-ET cycles [39]. One such factor may be blood flow parameters in the uterus, which can affect implantation success. Inadequate blood flow, as indicated by uterine artery color Doppler measurements, may lead to implantation failure in patients with unsuccessful IVF outcomes. Adequate vascular supply is essential for various IVF-related processes, including endometrial growth and embryo implantation [40].
Studies have demonstrated that the PI and RI of the uterine artery were significantly lower in women with successful IVF-ET cycles compared to those with unsuccessful cycles [41,42]. Our study yielded similar results [41-43]. Ng et al. [28] found that administering a single 20 mg dose of nifedipine 30 minutes before ET did not improve the IR or CPR in infertility treatment. Similarly, our study did not demonstrate a statistically significant difference in these outcomes.
The present results indicate decreases in the PI and RI of the uterine artery after approximately 14 to 20 days of amlodipine administration. This conclusion is supported by consistent pre- and post-intervention measurements. Participants were carefully selected to minimize variability, excluding those with conditions or medications that could influence PI or RI. To increase reliability, the study was performed in a controlled clinical setting to minimize external variables. Before initiating amlodipine administration, baseline values for PI and RI were established for each participant. A control group not receiving amlodipine was included to compare natural variations in PI and RI with those observed among medication recipients. The same protocols for measurement timing and data handling were applied to all participants, ensuring consistency and reliability. Participants were also monitored regarding additional factors that could affect PI and RI, such as diet, activity levels, and other medications, and these were controlled or accounted for in the analysis. Within its pharmacological class, amlodipine is notable for demonstrating prolonged efficacy by inhibiting voltage-dependent L-type calcium channels, thereby interrupting initial calcium influx. With a half-life of 30 to 50 hours, amlodipine enables convenient once-daily dosing, in contrast to shorter-acting alternatives like nifedipine [22]. Due to its long half-life, amlodipine is typically taken once daily, with effects lasting for 24 hours or more. Steady-state plasma levels are achieved after 7 to 8 days of daily dosing [44]. Therefore, given the mechanism of action and the timing of administration, we anticipated that the reduction in PI and RI of the uterine artery should be observable within 14 to 20 days of amlodipine use, a sufficient period to observe the drug’s stable effects. The objectives of the present study were to assess the impact of amlodipine on the PI and RI of the uterine artery and to improve ET outcomes by increasing circulation through blood vessel relaxation. Our findings demonstrate potential as a therapeutic approach in women experiencing implantation failure, meriting additional research. By providing a clear path, this study lays the groundwork for subsequent exploration of amlodipine in ET treatments. Increasing the sample size and potentially examining alternative medication protocols may be beneficial.
In conclusion, this study is the first to examine the use of amlodipine to correct uterine artery resistance in women with prior implantation failure, with the aim of guiding future research. However, our findings suggest that administering amlodipine before FET may not significantly improve clinical pregnancy outcomes for women with a history of FET cycle implantation failure. Further research is needed to explore the clinical potential of amlodipine, and clinical trials with larger sample sizes should be conducted.
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