Introduction
Polycystic ovary syndrome (PCOS) is a leading cause of anovulatory infertility in women of reproductive age and is characterized as a lifelong multigenic endocrinopathy [1]. According to the Rotterdam diagnostic criteria, a diagnosis of PCOS requires the presence of at least two of the following features: clinical or biochemical hyperandrogenism, ovarian dysfunction (evidenced by oligo- or anovulatory menstrual cycles), and polycystic ovarian morphology [2].
Clomiphene citrate (CC) is the first-line treatment for inducing ovulation in women with PCOS who are attempting to conceive [3]. However, about 15%–40% of these women show resistance to CC, characterized by the failure to ovulate after taking 150 mg daily for 5 days over three consecutive cycles [4].
The exact mechanisms and causes of PCOS remain unclear. Although PCOS is generally regarded as a hormone-related disorder, recent evidence suggests that oxidative stress (OS) and chronic low-grade inflammation are significant contributors to the development of PCOS and its associated metabolic conditions [5]. OS is implicated in disrupted ovarian steroidogenesis, which leads to increased androgen production and infertility [6]. OS is often assessed by measuring circulating markers such as malondialdehyde (MDA), indicative of oxidative damage [5]. Evidence of increased OS has been recognized in women with infertility and PCOS. Additionally, OS plays a crucial role in CC resistance [7].
Cytokines have been shown to play a direct role in regulating the complex balance of the hypothalamo-pituitary-ovarian axis and in maintaining normal ovarian and menstrual cycles [7]. The imbalance of cytokines, a key feature of chronic inflammation in PCOS, may also contribute to the diminished ovarian response to CC therapy [7]. In women with PCOS, high levels of tumor necrosis factor-α (TNF-α) in follicular fluid are inversely related to estradiol (E2) levels, indicating potentially poor oocyte and embryo quality [8]. Chronic low-grade inflammation and OS are considered the primary pathways in the pathogenesis of PCOS [9], suggesting that targeting these pathways with anticytokine and antioxidant agents could offer alternative treatment options [10].
Pentoxifylline (PTX) acts as a dual inhibitor of TNF-α production and xanthine oxidase, which may reduce OS and inflammation in a murine model of hyperandrogenism-induced PCOS [11]. PTX promotes normal folliculogenesis and promotes fertility through its antioxidant effects [11]. Additionally, it has demonstrated positive effects on type 1 diabetes by reducing OS markers while increasing antioxidant capacity [12]. Animal studies have demonstrated that PTX normalizes TNF-α levels, preserving ovarian steroid production and promoting regular follicle development [11]. Clinical trials have shown that a dosage of 400 mg of PTX taken twice daily can improve implantation and pregnancy rates in women with various infertility issues [13,14].
α-Lipoic acid (ALA) is recognized for its anti-inflammatory and antioxidant properties [15]. It may reduce inflammatory markers and enhance insulin sensitivity by promoting the translocation of glucose transporter type 4 to the cell membrane [16,17]. Additionally, ALA supplementation in PCOS patients could improve reproductive outcomes by positively influencing the physiological processes involved in oocyte recruitment, growth, and maturation [18].
In animal studies, adding ALA to the culture medium has been shown to enhance the development and growth of secondary preantral follicles [19]. Several clinical trials involving patients with PCOS undergoing in vitro fertilization (IVF) have suggested that ALA supplementation could improve both reproductive outcomes and metabolic profiles, potentially offering a long-term prevention strategy for PCOS [18,20]. This study aimed to assess the efficacy and safety of supplementing PTX and ALA to CCs to improve ovulation and implantation rates, thereby increasing the likelihood of conception in women with CC-resistant PCOS.
Methods
1. Study design
A prospective, randomized, controlled, open-label study was conducted from June 2022 to May 2023. This study included 120 women diagnosed with PCOS, who were recruited from the infertility unit of a university hospital and a licensed medical center specializing in infertility in Egypt.
2. Ethics
The study adhered to the Good Clinical Practice guidelines and the ethical principles outlined in the Declaration of Helsinki, as revised in 2013. Additionally, it followed the Consolidated Standards of Reporting Trials (CONSORT) guidelines and International Committee of Medical Journal Editors (ICMJE) recommendations. The protocol underwent revisions and received approval from the ethics committee for experimental and clinical studies at Ain Shams University (Approval number: ACUC-FP-ASU RHDIRB2020110301 REC#86). It was also registered with the Egyptian Ministry of Health (MOH). The ClinicalTrials.gov registration number is NCT05231980. Prior to participation, all patients were required to provide written informed consent.
3. Patient eligibility
Women with PCOS, diagnosed according to the revised 2003 Rotterdam consensus criteria [2], were included if they met the following criteria: women under 40 years old who had experienced infertility for more than 1 year and had failed to ovulate during the previous three uninterrupted cycles of CC at 150 mg/day for 5 days per cycle. The exclusion criteria included patients with diabetes mellitus, hypertension, liver or kidney dysfunction, heart disease, persistent hyperprolactinemia, thyroid dysfunction, acute myocardial infarction, severe coronary artery disease, hemorrhage, or peptic ulcers. Patients had undergone gonadotropin induction. Patients had been on vitamins and antioxidant supplements in the last 3 months.
4. Treatment intervention
All patients underwent eligibility screening. A total of 120 eligible patients were randomized into four groups. Randomization was achieved using a random sample allocator software available at https://www.graphpad.com/quickcalcs/randomize2/, assigning patients to one of the four groups in a 1:1:1:1 ratio. The study's principal investigator conducted this process.
Group 1, the control group, consisted of 30 patients who received 150 mg of CC (Clomid; Sanofi Aventis) for 5 days, starting on the third day of their menstrual cycle. Group 2 received 400 mg of PTX (Trental; Sanofi Aventis) twice daily, in addition to standard therapy. Group 3 was administered 600 mg of ALA (thiotacid; Eva Pharma) twice daily, also alongside standard therapy. Group 4 received both 400 mg of PTX and 600 mg of ALA twice daily, in addition to standard therapy. PTX and ALA treatment commenced 4 weeks prior to the start of induction cycles and continued throughout the 3-month follow-up period, which covered three induction cycles. All medications were discontinued upon confirmation of pregnancy.
The doses of PTX [13,14] and ALA [21] were chosen based on previous research and the availability of commercial products. A clinical pharmacist monitored all groups weekly for any adverse or side effects. Patients received education on the potential side effects of CC, PTX, and ALA, and were instructed to report any occurrences. Side effects were evaluated using an objective scale: 0 (no side effects), 1 (mild side effects that do not impact lifestyle), 2 (severe side effects, manageable with other methods), 3 (severe, unmanageable side effects, yet participants consent to continue the medication), and 4 (severe, unmanageable side effects leading participants to discontinue the medication) [22].
The incidence of symptoms potentially indicative of ovarian hyperstimulation syndrome (OHSS), including abdominal bloating, discomfort, significant weight gain (up to 10 to 15 pounds of fluid), nausea, constipation, and enlarged ovaries, was closely monitored. A complete history, encompassing patient demographics, baseline characteristics, and laboratory data, was collected from each patient. Additionally, all patients underwent comprehensive clinical testing prior to participation.
5. Laboratory measurements and ultrasonography
Five milliliters of venous blood were collected from each participant through antecubital venipuncture, transferred to a plain test tube, and centrifuged at 3,000 rpm for 10 minutes. The separated sera were then stored at –80 °C until analysis of baseline parameters and routine laboratory results. Serum levels of TNF-α were measured at the beginning and end of the study using a double-antibody sandwich enzyme-linked immunosorbent assay provided by Sun Red Biological Technology Co. Ltd. MDA, a marker of OS, was assessed at baseline and at the conclusion of the study using the method previously described by Draper and Hadley [23].
At baseline, we collected demographic and clinical data for all patients. Ultrasonography (US), along with power and color Doppler analysis, was conducted. Patients underwent a transvaginal US examination in the lithotomy position using a Voluson E6 (GE Healthcare) machine equipped with a 6.5 MHz transvaginal transducer. We then positioned a 2-mm range gate across the vessel to ensure the Doppler beam was nearly perpendicular to the vessel, with an angle close to 0°. To eliminate low-frequency signals from vessel wall movements, a 50–100 Hz filter was used.
The pulsatility index (PI) was electronically calculated using power and color Doppler from three consecutive waveforms of satisfactory quality. The ratio of peak systolic flow to the lowest diastolic flow (S/D ratio) was also recorded. To ensure consistency, all scans were conducted by the same operator to minimize variability in observations between different individuals.
The expected time for ovulation was determined through folliculometry, beginning on day 9. Women were monitored every other day until the mature follicle reached a size of 18 to 22 mm. They were advised to have intercourse every other day for 1 week and were then left to ovulate spontaneously without the administration of human chorionic gonadotropin (HCG). Serum progesterone (P21) levels were measured 8 days after the mature follicle was detected to confirm ovulation.
The PI of uterine blood flow was automatically calculated, and endometrial thickness (ET) was assessed on the day a follicle measuring 18 to 22 mm was detected [22]. For measurements of the uterine artery, color flow images facilitated the identification of the ascending branches, which were sampled laterally to the cervix in the sagittal plane. The number of mature follicle (NMF), mean follicular diameter (MFD), and ET were recorded for each menstrual cycle.
Ovulation was confirmed through US (corpus luteum) and by measuring serum midluteal P21 levels, which were greater than 5 ng/mL on day 21 of the menstrual cycle. Serum HCG levels were assessed 2 weeks following the absence of menstruation to diagnose pregnancy. Pregnant women were instructed to discontinue all medications. Patients lacking any dominant follicles were started on a new ovulation induction cycle the subsequent month. The primary outcome measure was the cumulative ovulation rate, defined as the proportion of cycles in which ovulation occurred throughout the entire follow-up period. Secondary outcome measures included NMF, ET, P21, MFD, PI, and pregnancy rates.
6. Statistical analysis
Data were collected, revised, coded, and entered into the SPSS ver. 27 (IBM Corp.). Quantitative data were presented as mean and standard deviation for parametric analyses and as median, minimum, maximum, and interquartile range for nonparametric analyses. Additionally, qualitative variables were presented as numbers and percentages.
The following tests were employed to compare categorical or nonparametric data: the Kruskal-Wallis U test, the chi-square test, or the Fisher exact test. Conversely, analysis of variance was utilized for comparing multiple groups that exhibited a normal distribution. The validity of both parametric and nonparametric tests of significance was confirmed using the Shapiro-Wilk test. Additionally, linear and logistic regression models were constructed using the selected variables as appropriate. All p-values were two-sided, and p-values less than 0.05 were considered statistically significant.
7. Sample size calculation
The sample size for our randomized controlled study was determined by comparing the ovulation rates (%) across four study groups. A previous publication reported an ovulation rate of 17.6% in the control group [24]. Anticipating moderate to high effects, we utilized an effect size (W=0.4) for the chi-square test, set the α level at 0.05, and established the degrees of freedom at 3. To achieve 90% power, the required sample size was 89 patients across the four groups. However, considering an expected dropout rate of 10% to 25%, we decided to recruit 120 patients, distributing them equally among the groups. The sample size calculations were performed using G*Power software ver. 3.1.2 (Heinrich Heine University) for MS Windows (Franz Faul, Kiel University) [25].
Results
1. Baseline characteristics
Participants’ baseline clinical characteristics are presented in Table 1. There were no significant differences among the four groups in terms of age, body mass index, duration of infertility, previous parity, or menstruation patterns. Additionally, baseline levels of several hormones, including follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroid stimulating hormone and prolactin, were measured and compared; Table 1 indicates that there were no significant differences in these hormone levels among the groups. All participants were free of comorbidities and were not taking any medications prior to their inclusion in the trial. A CONSORT diagram of the study groups is shown in Figure 1.
2. Evaluation of efficacy
The primary and secondary outcomes of ovulation induction cycles are presented in Table 2. The statistical analysis included ovulation and pregnancy rates, MFD, ET, NMF, PI, and P21, revealing significant differences among the four groups. The cumulative ovulation rate was highest in the combined PTX-ALA group at 77% (n=23), followed by the PTX group at 70% (n=21), the ALA group at 40% (n=12), and the control group at 30% (n=9) (p=0.0003). The cumulative pregnancy rate was 40% (n=12) in the combined PTX-ALA group, 37% (n=11) in the PTX group, 10% (n=3) in the ALA group, and 3% (n=1) in the control group (p=0.0005).
Regarding ET, significant differences were observed among the four groups across the three visits (p<0.001), as detailed in Table 2. Post hoc analysis indicated that the control group exhibited a significantly lower ET compared to both the PTX group and the PTX-ALA group (p<0.001 for both). However, there was no significant difference in ET between the control group and the ALA group (p=0.886). Similarly, no significant difference was noted between the PTX group and the PTX-ALA group (p=0.826). Nonetheless, both these groups demonstrated significantly higher ET compared to the ALA group (p<0.001 for both), as indicated in Table 3.
For the MFD measurements, significant differences were observed among the four groups across the three visits, with p-values of less than 0.001 at the first and third visits, and p=0.01 at the second visit, as detailed in Table 2. Post hoc analysis revealed that the control group had a significantly lower MFD compared to both the PTX group and the PTX-ALA group (p<0.001 for both comparisons). However, there was no significant difference in MFD between the control group and the ALA group. Additionally, no statistically significant difference was found between the PTX group and the PTX-ALA group (p=0.831). Nevertheless, both the PTX and PTX-ALA groups showed a significant increase in MFD compared to the ALA group (p<0.001 for both), as shown in Table 3.
With respect to NMF, significant differences were observed among the four groups across the three visits (p<0.009 at the first visit and <0.001 at both the second and third visits), as detailed in Table 2. Post hoc analysis revealed that the control group exhibited a significantly lower mean NMF compared to both the PTX group and the PTX-ALA group (p<0.001 for both). However, there was no significant difference in NMF between the control group and the ALA group (p=0.721). While the PTX group and the PTX-ALA group showed no significant differences in NMF (p=0.94), both groups had significantly higher NMF levels than the ALA group (p=0.002 and p<0.001, respectively), as indicated in Table 3.
Regarding serum P21, significant differences were observed among the four groups at each of the three visits (p<0.001 for all visits), as indicated in Table 2. Post hoc analysis revealed that the control group had significantly lower P21 levels than both the PTX group and the PTX-ALA group (p<0.001 for both), with no significant difference observed when compared to the ALA group (p=0.671). Additionally, there was no significant difference between the PTX group and the PTX-ALA group (p=0.96). However, both these groups exhibited significantly higher P21 levels than the ALA group (p<0.001 for both), as shown in Table 3.
For the PI, significant differences were observed among the four groups across the three visits (p<0.001 for all visits), as detailed in Table 2. Post hoc analysis revealed that the control group exhibited a significantly lower PI than both the PTX group and the PTX-ALA group (p<0.001 for both); however, it did not differ significantly from the ALA group (p=0.247). Similarly, there was no significant difference between the PTX group and the PTX-ALA group (p=0.703); yet, both groups had significantly lower PI levels than the ALA group (p=0.002 and p<0.001, respectively), as shown in Table 3. Additionally, a subgroup analysis focusing on primary and secondary outcomes between group 2 (PTX) and group 4 (PTX-ALA) revealed no statistically significant differences in ovulation rate, pregnancy rate, and ET, as indicated in Table 4.
Side effects were categorized as no symptoms, mild, or severe, with no significant differences observed among the groups in either the occurrence of side effects or patient medication compliance, as detailed in Table 5. Similarly, there was no significant difference among the four groups in the incidence of common side effects such as vomiting, breast tenderness, gastric upset, and hot flushes, as presented in Table 6. No cases of OHSS were reported. A repeated measures analysis of covariance model, adjusted for pretreatment TNF-α and MDA serum levels, was employed to compare the pre- and post-treatment mean differences in TNF-α and MDA serum levels across all groups, as shown in Table 7.
Discussion
To the best of our knowledge, this study is the first randomized controlled trial to evaluate the effects of PTX and ALA on women with CC-resistant PCOS. The objective was to assess the safety and efficacy of PTX and ALA in improving the ovulation rate among these women. Our findings indicate that the groups treated solely with PTX experienced significant increases in both ovulation and pregnancy rates in women with CC-resistant PCOS.
In the present study, groups treated with PTX exhibited a significantly higher NMF compared to both the control group and the ALA group. Consistent with the findings of Rezvanfar et al. [11], our data demonstrate the positive impact of PTX on oocyte development and maturation, as evidenced by an increased ovulation rate. The beneficial effects observed can be attributed to several mechanisms: a reduction in TNF-α activity, which aids in reducing inflammation; the suppression of harmful free radical production in the ovary coupled with an enhancement of overall antioxidant capacity; and the maintenance of adequate E2 levels, which leads to a reduction in follicular atresia [11].
Vitale and colleagues demonstrated that administering PTX during ovarian stimulation could enhance IVF outcomes by not only generating a thicker endometrial lining, but also by producing a greater number of oocytes with better oocyte, zygote, and embryo quality and higher serum E2 concentrations [26]. This improvement can be attributed to the effects of PTX on cyclic adenosine monophosphate, which amplifies the actions of FSH and LH, the gonadotropins essential for follicular maturation and ovulation [25].
Letur-Konirsch and Delanian [13] reported that in women with premature ovarian failure, the combination of vitamin E and PTX improved embryo implantation and ongoing pregnancy rates through an antioxidant mechanism. Another study demonstrated that supplementation with PTX and vitamin E for 6 months enhanced implantation and pregnancy rates in patients with thin endometria undergoing IVF with oocyte donation [14].
The administration of ALA led to increased ovulation and pregnancy rates compared to the control group. However, the differences between the ALA group and the control group were not statistically significant. This observation aligns with a study by Rago et al. [18], which also found no significant difference in pregnancy rates, although there was a trend toward higher rates in patients receiving both myo-inositol and ALA compared to those receiving only myo-inositol.
These findings confirm that ALA had beneficial effects on metabolic outcomes rather than reproductive outcomes in patients with PCOS [27]. In contrast to our results, some studies have suggested that ALA might significantly increase pregnancy and ovulation rates [18,20]. This discrepancy may be explained by the fact that those studies focused on non-CC-resistant patients.
The beneficial effects of antioxidants were examined in CC-resistant PCOS. N-acetylcysteine produced a significant pregnancy rate of 27% and an ovulation rate of 42% [28]. In another study, L-carnitine was associated with a pregnancy rate of approximately 57.1% and an ovulation rate of approximately 71.4% [29]. These findings are consistent with our results, which showed a pregnancy rate of 40% and an ovulation rate of 77%.
Regarding serum P21 levels, both the PTX group and the PTX-ALA group exhibited significantly higher concentrations compared to those in the ALA group and the control group. This difference could contribute to the increased pregnancy rates observed in the PTX and PTX-ALA groups. This observation aligns with a clinical study that indicated an increase in P21 levels among PCOS patients, which led to improved pregnancy rates [30].
ET was significantly higher in the PTX group and the PTX-ALA group compared to the control group and the ALA group. The increased ET in response to PTX could be another potential reason for the higher pregnancy rates observed in the PTX group. This improvement may be attributed to the hormonal corrective actions of PTX and its antioxidant effects [12]. Previous studies also have shown that PTX downregulates cytokines such as interleukin 6 and TNF-α and decreases the anti-proliferative effect induced by these cytokines [31].
Regarding the PI of the uterine artery, groups 2 (PTX) and 4 (PTX-ALA) exhibited a significantly lower PI compared to group 1 (control) and group 3 (ALA). A decrease in the PI enhances uterine blood flow, which in turn improves ET and potentially increases the pregnancy rate. This finding aligns with other research indicating that a significant reduction in the PI is associated with higher pregnancy and ovulation rates [22,32]. This might explain the positive impact of PTX on blood flow in the subendometrial and endometrial regions, potentially enhancing endometrial receptivity during the peri-implantation period. Additionally, similar outcomes have been observed in animal studies, attributed to the antioxidant and antiapoptotic effects of PTX [11].
In this study, both PTX and ALA significantly reduced serum MDA levels, corroborating findings from a previous study where PTX administration led to a notable decrease in MDA levels [33]. Panti et al. [34] reported a significant reduction in MDA, as well as a significant increase in the pregnancy rate in PCOS patients. Oral et al. [35] reported that pregnancy rates decreased with increasing MDA levels of patients in their study.
Regarding TNF-α, Rezvanfar et al. [11] reported that significantly reducing TNF-α preserved ovarian function and maintained a normal ovulation rate in rats with hyperandrogenism-induced PCOS induced using PTX. Similarly, Mohammadi et al. [36] observed a significant decrease in TNF-α levels, which corresponded with improved ovulation in patients with PCOS. Pawelczak et al. [37] reported that serum TNF-α levels may be elevated in adolescent patients with PCOS. These high levels might contribute to the potential development of infertility [37].
Another study by Xiong et al. [38] reported that patients with PCOS exhibited significantly elevated levels of inflammatory cytokines, particularly TNF-α. This increase in TNF-α was linked to the induction of ovarian anovulation through apoptosis affecting granular and theca cells. As a result, dominant follicles failed to develop, and heightened TNF-α expression may be associated with implantation failure [39]. Both PTX and ALA were well-tolerated by most patients and did not lead to any severe adverse effects or exacerbate the adverse effects of other medications used in our study.
There were some limitations in our study. First, a placebo was not employed (open-label design) due to the complexity of involving multiple drugs and four distinct groups, which made the preparation of several placebos impractical. However, the open-label nature of the study may have introduced observer and participant biases. For instance, knowing which treatment was administered could affect how participants report subjective outcomes like side effects or satisfaction. Despite these concerns, similar results were found in both open-label studies [18,40,41] and double-blinded studies [29,42], suggesting that the lack of blinding did not impact our study results.
Second, the study provides valuable insights into the short-term effects of the combined therapy; however, its long-term efficacy and safety remain uncertain. The follow-up period, limited to three cycles, effectively captured short-term outcomes such as ovulation and early pregnancy rates. Yet, it failed to explore long-term impacts, including live birth rates, ongoing pregnancies, or the recurrence of CC resistance after therapy discontinuation. These factors are essential for assessing the overall success and sustainability of the treatment. The limited duration of the follow-up can be attributed to the nature of most clinical trials involving CC-resistant patients, which typically do not extend beyond three cycles. This restriction is due to the potential for side effects when treatment is prolonged beyond this duration in patients who have previously received CC.
Additionally, this combination therapy served as a preliminary trial before considering options such as gonadotrophin therapy, laparoscopic ovarian drilling, or advancing to IVF. Consequently, patients were not required to endure lengthy waiting periods at this stage. Third, this study specifically focused on women resistant to CC, who experience higher rates of ovulation and pregnancy failure compared to those sensitive to CC. This may restrict the generalizability of the findings to the wider PCOS population.
The clinical implications of this study are significant, as it demonstrates marked increases in ovulation and pregnancy rates in the PTX plus CC group. This finding may support the potential of PTX as an adjunct therapy in women with CC-resistant PCOS before considering more invasive procedures such as gonadotropin therapy or laparoscopic ovarian drilling. Both PTX and ALA target OS and chronic inflammation, which are key factors contributing to CC resistance.
Through this mechanism, it may improve not only ovulation but also overall reproductive outcomes. In the PTX group, there was an improvement in endometrial receptivity and blood flow, as indicated by a decreased PI. This enhancement can directly benefit implantation rates, making this combination valuable for improving fertility outcomes. Compared to gonadotropin therapy or IVF, combining PTX with CC offers a non-invasive and potentially cost-effective option for patients with CC-resistant PCOS.
In conclusion, the findings of this study affirm the therapeutic advantages of using PTX as an adjunctive treatment for CC, demonstrated by the significant improvements in ovulation and pregnancy rates. Therefore, incorporating PTX as an additional therapy is advisable for managing women with CC-resistant PCOS. The notable efficacy of PTX may be due to its antioxidant and anti-inflammatory properties. Given these benefits, PTX could potentially become the preferred adjuvant therapy for CC, and should be considered before initiating gonadotrophin therapy or laparoscopic ovarian drilling in patients with CC-resistant PCOS.
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