Abstract
Objective
To optimize ovulation induction protocols for infertile women with PCOS, ovulation effect and adverse reactions of different doses of letrozole (2.5 vs 5.0 mg) combined sequentially HMG therapy were compared in infertility PCOS patients.
Methods
This open-label randomized controlled trial (RCT) included 174 infertile women aged 18–40 who met the Rotterdam criteria for PCOS at the Wuhan Union Hospital of China from May 2021 to January 2022. They were randomly assigned at a 1:1 ratio to 2.5 mg LE or 5.0 mg LE on cycle days 3–7 with sequential HMG injections (n = 87 for each).
Results
There is no difference in ovulation rate between LE (2.5 mg) + HMG group and LE (5.0 mg) + HMG group in infertile women with PCOS (85.1 vs 85.1%). The ongoing pregnancy rate was no different between the two groups (33.3 vs 25.3%). The percentage of type B endometrial tissues on HCG injection day was higher in the LE (2.5 mg) + HMG group (88.5% vs 69.0%). The monofollicular development rate was significantly higher in the LE (2.5 mg) + HMG group (67.8% vs. 46.0%).
Conclusions
Application of 5.0 mg LE followed with HMG does not improve the pregnancy rate compared to 2.5 mg LE in infertile women with PCOS. An increased dose of LE to 5.0 mg may increase the risks of OHSS and multiple pregnancies. Therapy of LE (2.5 mg) + HMG may be a more beneficial and optimal treatment protocol for improving endometrial receptivity and promoting mono-follicle development for patients with PCOS.
Keywords: Human menopausal gonadotropin, Letrozole, Infertility, Ovulation induction, Polycystic ovary syndrome, Different doses
Introduction
Polycystic ovary syndrome (PCOS) is a complex reproductive-metabolic disorder characterized by oligo/anovulation, elevated androgen levels, and polycystic ovaries. PCOS is the most prevalent cause of anovulatory infertility, with an incidence rate of 5 to 10% in reproductive-age women [1]. Ovulation induction therapy is a mainstay of PCOS treatment for infertility. The traditional therapy applied clomiphene citrate (CC) to induce ovulation in this condition. However, approximately 25% of women with PCOS will not respond at all to CC. CC also has an anti-estrogenic effect on endometrial development and cervical mucus production, which have been implicated in a low pregnancy rate.
Alternative treatments for CC, such as letrozole (LE), have attracted attention in the past few years. LE is a third-generation, potent, and selective aromatase inhibitor that blocks estrogen synthesis through the direct suppression of their conversion to androgens. It works by inhibiting estrogen production, reducing the negative feedback on the hypothalamus-pituitary axis, and subsequently resulting in increasing secretion of follicle-stimulating hormone (FSH), which stimulates ovarian follicle development and maturation. Several meta-analyses published in recent years found that the pregnancy and live birth rates of infertile PCOS patients undergoing LE treatment were significantly higher than those receiving clomiphene citrate (CC) [2–5]. According to the European Society of Human Reproduction and Embryology guideline (ESHRE 2018) [6], LE is recommended as the preferred first-line treatment for infertile women with PCOS. However, the ovulation and pregnancy rate of LE alone are not high enough.
Human menopausal gonadotropin (HMG) contains both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) [7]. It is one of the most commonly used drugs for ovulation induction treatment [7]. Since HMG has a strong pharmacological effect and PCOS patients are particularly susceptible to gonadotropins [7], ovarian hyperstimulation syndrome (OHSS) and multiple pregnancies continue to be major drawbacks of using HMG alone [8, 9]. At present, various ovulation drugs have their own advantages and disadvantages. There is no standard treatment for ovulation induction for infertile PCOS patients.
HMG is frequently used with LE and other ovulation induction drugs to limit the incidence of the aforementioned adverse events while also controlling costs [6, 10, 11]. In a previous study, the application of LE followed with HMG was proven to be more effective in improving the ovulation rate and live birth rate than LE alone [12, 13], while there was no statistical difference in OHSS, multiple pregnancies, miscarriages, and other minor drug side effects. The efficacy of different dosages of LE in ovulation induction is critical for clinicians to develop effective treatment strategies. It has been confirmed that LE at 5 mg/day was shown to be more effective than 2.5 mg/day [14], while 7.5 mg/day had no advantage over 5 mg/day [15]. Clinical research on the therapeutic impact of different dosages of LE combined sequentially with HMG in infertile women with PCOS is currently insufficient. To address this problem, we conducted a randomized controlled clinical trial to compare the ovulation effect and adverse reactions of different doses of LE (2.5 mg vs 5.0 mg) combined HMG therapy. The purpose of this study is to find an appropriate ovulation induction protocol concerning PCOS with anovulation infertility.
Materials and methods
Participants
The open-label randomized controlled clinical trial was conducted at the Wuhan Union Hospital and enrolled 174 participants who fulfilled the inclusion criteria for this study and desired to become pregnant. The first participant was enrolled in May 2021, and the last one in January 2022.
The inclusion criteria were as follows: a. PCOS patients diagnosed according to the Rotterdam criteria in 2003 [16] (diagnosis was made if two of the three factors were noted: ovulatory dysfunction, hyperandrogenism (on the basis of hirsutism or an elevated testosterone level), and polycystic ovary(ies) under gynecologic ultrasound); b. age 18–40 years, normal sexual life without the use of contraceptive techniques, and the duration of infertility of at least 1 year; c. hysterosalpingography revealed a normal shape of the uterus, and at least one side of the fallopian tube was unobstructed; d. sperm analysis of the spouse was normal; e. organ functionality was normal and without any endocrine diseases, such as hypopituitarism or hyperthyroidism.
The exclusion criteria were as follows: a. women with known allergies or allergies to the relevant drugs; b. women with cardiovascular diseases, systemic complications, and obvious abnormality or impairment of the liver and kidney functions; c. pregnancy or lactation; d. past uterine surgery or uterine anomalies, e. past exposure to teratogenic poisons and radiation.
All participants were expected to read the informed consent form, voluntarily agree to participate in this study, and provide a signed informed consent form to confirm their participation in the study.
Interventions
All participants received ethinylestradiol and cyproterone acetate tablets (Diane-35 Schering, Berlin, Germany) for 1–3 months as a pre-treatment before administration of ovulation-inducing drugs. The baseline characteristics evaluation was performed on day 3 of the menstrual cycle, and all patients were treated by ovulation induction. The eligible patients were randomly allocated into two groups (group 2.5 mg LE + HMG and group 5.0 mg LE + HMG) in a 1:1 ratio, using a random number table generated by the computer. Participants in group 2.5 mg LE + HMG were prescribed 2.5 mg LE (trade name: Fu Rui, Jiangsu Hengrui Medicine Co., Ltd.) on cycle days 3–7 with a sequential therapy of 75-IU HMG (Lizhu Pharmaceutical Factory) daily from cycle day 8 until the presence of at least one mature follicle, defined as having a diameter of 18 mm or greater, was confirmed by ultrasound. Meanwhile, participants in group 5.0 mg LE + HMG received 5.0 mg of LE daily on cycle days 3–7 with a sequential therapy of 75 IU HMG (Lizhu Pharmaceutical Factory) from cycle day 8 every day until at least one mature follicle was visible on ultrasound. HMG duration (3–10 days) depends on the subject’s developmental status of follicles (HMG can be used for up to 10 days). The follicular development was monitored through transvaginal ultrasound in all patients starting from day 11 of the cycle. The record of the number/size of follicles and the endometrial thickness was performed during scanning. Human chorionic gonadotropin (HCG) was administered to induce ovulation when at least one follicle reached a diameter of ≥ 18 mm. The blood was collected to determine the level of sex hormones (progesterone, estradiol and LH) at the same time, followed by sexual intercourse 24–36 h later. Vaginal B-ultrasound was performed 48 h after HCG injection to monitor ovulation. Luteal support was provided after ovulation through oral administration of 20 mg dydrogesterone (trade name: DaFuTong, Abbott Healthcare Products B.V.) each day for 2 weeks. The plasma β-HCG levels were measured 2 weeks later to determine the conception status. B-ultrasound was performed 4 weeks later to diagnose clinical pregnancy. Ongoing pregnancy was defined by a normal fetal nuchal translucency (NT) test result, which served as the endpoint of the study. Patients in each study arm received a single cycle of treatment. Any adverse reactions occurring during the treatment of all participants were recorded. All participants had their endometrial thickness and type monitored and recorded through transvaginal ultrasound during the study process. This study evaluated endometrial ultrasound morphology based on the classification criteria proposed by Gonen in 1990 [17], categorizing the endometrium into types A, B, and C.
Outcome measures
The primary outcome was the ovulation rate. Secondary outcomes included the following: conception, clinical pregnancy, ongoing pregnancy (fetal nuchal translucency (NT) check was performed to diagnose ongoing pregnancy), singleton pregnancy, multiple pregnancies (the singleton and multiple pregnancy samples were sourced from the clinical pregnancy samples, but not from the ongoing pregnancy samples), pregnancy loss (including biochemical, miscarriage, or ectopic cases), and drug-related side effects and adverse reactions.
Sample size
The sample size was calculated by the G*Power 3.1.9.7 software, and the enrolled sample size was 74 subjects in each group, as per a preliminary study conducted previously [18, 19] and based on the sample size calculation formula with a two-sided 0.05 significance level and 0.95 statistical power by using Pearson’s Chi-squared test. In two prospective clinical randomized trials [18, 19], the absolute difference in the ovulation rate was 30.3%, in which the former study showed that 2.5 mg LE presented with a 41.2% ovulation rate, and the latter showed that 5.0 mg LE presented with 71.5% ovulation rate. After applying the correction allowed for a dropout rate of 19%, the final sample size was 87 (1.19 × 73) per group, with due consideration of the noncompliance and the rate of loss to follow-up.
Randomization
The present study had an open-label, parallel-group, randomized, controlled design. The enrolled participants were assigned into two groups in a 1:1 ratio using a random number table generated by the IBM SPSS Statistics software: group 2.5 mg LE + HMG and group 5.0 mg LE + HMG. Doctors and patients were both aware of the treatment group allocations, but the sonographers, statisticians, and outcome assessors were blinded to the study group assignments.
Statistical analyses
For this RCT, the primary outcome was analyzed using an intention-to-treat (ITT) and a per-protocol (PP) method. ITT analysis included every subject who was randomized according to the randomized treatment assignment, whereas the PP analysis only included participants who finished the treatment and the subsequent follow-up. Participants lost to follow-up were considered to have not reported any ongoing pregnancy in the ITT analysis.
The statistical analyses were performed using the IBM SPSS Statistics (25.0), R (3.6.3), and Python (3.7). Qualitative (categorical) variables were presented as the number of cases with percentages (%), and the differences between the two groups were compared by using the Chi-squared test and Fisher’s exact test (two-sided). Statistical differences were considered significant at P < 0.05. For quantitative (numerical) variables, all data were analyzed for normal distribution by performing the kurtosis and skewness test for normality. In the case of normal data, two independent-sample Student’s t-test was applied, and the mean ± standard deviation was reported; otherwise, the Mann–Whitney U-test was performed, and the data was presented in the median and interquartile range. The differences were considered to be statistically significant at P < 0.05.
A subgroup analysis was applied when assessing fecundity and cycle characteristics among patients who ovulated and who conceived. Stratification analyses on the body mass index (BMI), insulin resistance (IR), and waist-hip ratio (WHR) were conducted to control the confounding factors, respectively, considering that the factors could be associated with infertility treatment success. Strata 1 was BMI < 18, strata 2 was 18 ≤ BMI < 24, strata 3 was 24 ≤ BMI < 28, strata 4 was BMI ≥ 28, strata 5 was WHR ≤ 0.85, strata 6 was WHR > 0.85, strata 7 was IR YES, and strata 8 was IR NO (patients with insulin resistance, defined as the value of homeostasis model assessment of insulin resistance (HOMA-IR) ≥ 2.69) [20].
Results
Participant flow
According to the inclusion/exclusion criteria, 174 participants were allocated from 188 invited patients who were preliminarily eligible. Then, 87 subjects were randomly assigned to either the sequential LE (2.5 mg) + HMG group or the sequential LE (5.0 mg) + HMG group. The treatment regimens were completed by all 174 participants, except for a participant in the 5.0 mg group who was lost to follow-up after clinical pregnancy confirmed by B-scan ultrasonography. In the intention-to-treat (ITT) analysis, 174 patients were included, while 173 patients were included in the per-protocol (PP) analysis (Fig. 1).
Fig. 1.
CONSORT diagram illustrating the participant’s flow at each stage of the study
Baseline data
The baseline characteristic of LE (2.5 mg) + HMG group and LE (5.0 mg) + HMG group were similar, including biometric features, fertility history, metabolic status, sex hormone level, ultrasonographic findings and clinical features (Table 1). No statistical difference was observed from all baseline data between the two groups.
Table 1.
Baseline characteristic of the study participants
| Characteristic | 2.5 mg + HMG (n = 87) | 5.0 mg + HMG (n = 87) |
|---|---|---|
| Biometric features | ||
| Age, years | 28.0 [26.0, 30.0] | 28.0 [26.0, 30.0] |
| Body fat percentage, % | 33.7 [30.4, 37.8] | 35.5 [31.4, 38.8] |
| Visceral fat area, cm2 | 88.8 [68.3, 122.2] | 106.1 [78.6, 129.1] |
| Waist-hip ratio | 0.87 [0.84, 0.91] | 0.88 [0.86, 0.91] |
| BMI, kg/m2 | 23.20 [20.89, 25.82] | 24.12 [21.9, 27.03] |
| Weight, kg | 59.1 [53.9, 66.8] | 61.6 [55.4, 68.5] |
| Fertility history | ||
| Primary infertility | 68(78.16%) | 70(80.46%) |
| Infertility duration | 2.0 [1.0,3.0] | 2.0 [1.0, 3.0] |
| Metabolic status | ||
| Fasting insulin, mIU/L | 12.64 [9.55, 16.96] | 13.48 [9.0, 21.62] |
| Fasting glucose, mmol/L | 5.2 [4.89, 5.7] | 5.2 [4.82, 5.6] |
| HOMA-IR | 3.03 [2.14, 4.16] | 3.03 [2.15, 4.97] |
| Sex hormone | ||
| Testosterone, nmol/L | 1.46 [0.99, 2.07] | 1.52 [1.12, 2.09] |
| PRL, IU/L | 20.24 [15.49, 27.62] | 19.53 [13.69, 24.42] |
| FSH, ng/mL | 5.86 [5.03, 7.09] | 6.27 [5.19, 7.53] |
| LH, IU/L | 8.52 [5.89, 12.93] | 10.02 [6.41, 14.23] |
| LH/FSH | 1.45 [0.98, 2.17] | 1.74 [1.14, 2.23] |
| Ultrasonographic findings and clinical features | ||
| Types of endometrial pattern | C (100%) | C (97.70%) |
| Endometrial thickness (mm) | 4.0 (4.0, 5.0) | 4.0 (3.0,5.0) |
Values are presented as n (%) or median (interquartile range). P-values were calculated by Chi-squared test or Fisher’s exact test for qualitative data and t-test or by Mann–Whitney U-test for qualitative data. All P-values were insignificant. BMI, body mass index; PRL, prolactin; HOMA-IR, homeostasis model assessment of insulin resistance [20]
Primary outcome
We contrasted reproductive outcomes, including ovulation and fecundity, between the two groups of patients (Table 2). There is no difference in ovulation rate between the LE (2.5 mg) + HMG group and the LE (5.0 mg) + HMG group (P = 1.000), and the per-protocol (PP) analysis revealed similar results (P = 0.974) (Table 2). A forest plot was utilized in the study to evaluate the ovulation rates for each stratum, as detailed in Table 3. The analyses in this study were stratified based on BMI, waist-to-hip ratio, and insulin resistance. In each stratum, there was no statistically significant difference (Table 3).
Table 2.
Reproductive outcomes
| Reproductive outcomes | 2.5 mg + HMG (n = 87) | 5.0 mg + HMG (n = 87) | RR (95%CI) | Absolute difference (95% CI) | P-value |
|---|---|---|---|---|---|
| Primary outcome | |||||
| Intention-to-treat analysisa | (n = 87) | (n = 87) | |||
| Ovulation | 74/87 (85.1%) | 74/87 (85.1%) | 1.00 (0.88,1.13) | 0.0% (−10.8%−10.8%) | 1.000 |
| Ongoing pregnancy | 29/87 (33.3%) | 22/87 (25.3%) | 1.21 (0.77,1.90) | 5.8%(−7.9%−19.1%) | 0.410 |
| Per-protocol analysisb | (n = 87) | (n = 86) | |||
| Ovulation | 74/87 (85.1%) | 73/86 (84.9%) | 1.00 (0.88,1.14) | 0.2% (−10.7%−11.0%) | 0.974 |
| Ongoing pregnancy | 29/87 (33.3%) | 22/86 (25.6%) | 1.19 (0.76,1.88) | 5.4% (−8.2%−18.8%) | 0.439 |
| Secondary outcome | |||||
| Overall reproductive outcomes | (n = 87) | (n = 87) | |||
| Conception | 36/87 (41.4%) | 28/87 (32.2%) | 1.29 (0.87,1.91) | 9.2% (−5.1%−23.0%) | 0.208 |
| Clinical pregnancy | 34/87 (39.1%) | 25/87 (28.7%) | 1.26 (0.84,1.90) | 8.1% (−6.1%−27.7%) | 0.266 |
| Singleton pregnancy | 30/87 (34.5%) | 20/87 (23.0%) | 1.41 (0.89,2.23) | 9.2% (−4.4%−22.3%) | 0.138 |
| Multiple pregnancy | 4/87 (4.6%) | 5/87 (5.7%) | 0.80 (0.22,2.88) | 1.2% (−6.3%−8.7%) | 1.000c |
| Pregnancy loss | 6/87 (6.9%) | 6/87 (6.9%) | 1.00 (0.34,2.98) | 0.0% (−8.2%−8.2%) | 1.000c |
| Biochemical pregnancy | 2/87 (2.3%) | 1/87 (1.1%) | |||
| Early miscarriage | 4/87 (4.6%) | 3/87 (3.4%) | |||
| Ectopic gestation | 0/87 (0.0%) | 2/87 (2.3%) | |||
| Fecundity among those who ovulated | (n = 74) | (n = 74) | |||
| Conception | 36/74 (48.6%) | 28/74 (37.8%) | 1.29 (0.88,1.87) | 10.8% (−5.1%−25.9%) | 0.184 |
| Clinical pregnancy | 34/74 (45.9%) | 25/74 (33.8%) | 1.26 (0.85,1.86) | 9.5% (−6.3%−24.6%) | 0.242 |
| Singleton pregnancy | 31/74 (41.9%) | 20/74 (27.0%) | 1.41 (0.91,2.19) | 12.2% (−3.2%−26.8%) | 0.123 |
| Multiple pregnancy | 4/74 (5.4%) | 5/74 (6.8%) | 0.80 (0.22,2.86) | 1.4%(−7.2%−10.1%) | 1.000c |
| Ongoing pregnancy | 29/74 (39.2%) | 22/74(29.7%) | 1.21(0.78,1.87) | 6.8%(−8.6%−21.6%) | 0.391 |
| Pregnancy loss | 6/74 (8.1%) | 6/74(8.1%) | 1.00(0.34,2.96) | 0.0%(−9.5%−9.5%) | 1.000c |
| Pregnancy status of those who conceived | (n = 36) | (n = 28) | |||
| Clinical pregnancy | 34/36(94.4%) | 25/28(89.3%) | 0.98(0.88,1.09) | 2.0%(−12.7%−14.9%) | 0.709 |
| Singleton pregnancy | 31/36(86.1%) | 20/28(71.4%) | 1.10(0.88,1.39) | 7.5%(−11.0%−27.3%) | 0.428 |
| Multiple pregnancy | 4/36(11.1%) | 5/28(17.9%) | 0.62(0.18,2.10) | 0.4%(−16.7%−19.3%) | 0.488c |
| Ongoing pregnancy | 29/36(80.6%) | 22/28(78.5%) | 0.94(0.75,1.17) | 5.2%(−14.6%−23.0%) | 0.587 |
| Pregnancy loss | 6/36(16.7%) | 6/28(21.4%) | 0.78(0.28,2.15) | 4.8%(−14.2%−24.9%) | 0.750c |
a2.5 mg Letrozole + HMG, n = 87; 5.0 mg letrozole + HMG, n = 87;
b2.5 mg letrozole + HMG, n = 87; 5.0 mg letrozole + HMG, n = 86;
Fisher’s exact test
Table 3.
Stratification analysis
IR insulin resistance, WHR Waist-hip ratio. aFisher exact tes
Secondary outcomes
Of the 87 women in the LE (2.5 mg) + HMG group, 36 had a positive serum HCG test, 2 of which were biochemical pregnancies abortion, and 34 were diagnosed clinically pregnant (30 singletons and 4 multiples) by subsequent B-ultrasound examination. Considering the risks associated with multiple pregnancies in the later stages of gestation and the patient’s personal desire to undergo fetal reduction surgery, one patient had a first-trimester fetal reduction to reduce a quadruplet pregnancy to twins (ultrasound assessment of nuchal translucency (NT) was normal after fetal reduction). Then 3 missed miscarriages, with two cases occurring at 9 weeks of gestation and one at 10 weeks, while the other 29 were still pregnant following the NT scan. Two NT measurements in the LE (2.5 mg) + HMG group were abnormal, and all of them were isolated with increased NT with no other additional abnormalities.
Of the 87 women in the LE (5.0 mg) + HMG group, 28 were with a positive serum HCG test, 1 of which was biochemical pregnancy, 2 of which were diagnosed with ectopic gestation by subsequent B-ultrasound examination, and the remaining 25 were diagnosed clinically pregnant (20 singletons and 5 multiples). Considering the risks associated with multiple pregnancies in the later stages of gestation and the patient’s personal desire to undergo fetal reduction surgery, one patient received first-trimester fetal reduction to reduce a twin pregnancy to a singleton (ultrasound assessment of nuchal translucency was normal after fetal reduction). Then, 2 had a miscarriage, 1 was lost to follow-up after confirming a clinical pregnancy at 9 weeks of gestation, and the other 22 were still pregnant after NT measurements.
There was no statistically significant difference in conception (P = 0.208), clinical pregnancy (P = 0.266), singleton pregnancy (P = 0.138), ongoing pregnancy (P = 0.410) between the two groups (ITT analysis and PP analysis) (Table 2). There was also no difference between multiple pregnancies (P = 1.000) and pregnancy loss (P = 1.000) between the two groups. In women who have successfully ovulated, there is no significant difference in the improvement of subsequent pregnancy outcomes between the two treatment groups. Among those who conceived, none of these differences between the two groups were statistically significant.
The forest plot is shown in Table 3, although the difference did not reach statistical significance, the beneficial effect of ongoing pregnancy from LE (2.5 mg) + HMG treatment was seen in each strata. The comparable preceding but nonsignificant trend was probably attributed to the small sample size.
The endometrial morphology on HCG injection day was significantly different between the two groups (P = 0.003); a better result was found for LE (2.5 mg) + HMG group. The mono follicular development rate was significantly (P = 0.004) higher in the LE (2.5 mg) + HMG group than in the LE (5.0 mg) + HMG group (Table 4), but none of the other differences were statistically significant in sexual hormone levels, the dosage of gonadotropins, and endometrial thickness on hCG injection day between the two groups. Among those who had mature follicles, the number of cycles with one mature follicle was higher in the LE (2.5 mg) + HMG group than in the LE (5.0 mg) + HMG group (P = 0.002). The incidence of multi-follicular development is higher in LE (5.0 mg) + HMG group (28.4 vs. 55.4%, P = 0.001) among women who ovulated. There are no statistically significant differences in sexual hormone levels, the dosage of gonadotropins, and endometrial thickness on hCG injection day between the two groups.
Table 4.
Cycle characteristics
| Cycle characteristics | 2.5 mg + HMG | 5.0 mg + HMG | P-value |
|---|---|---|---|
| Overall characteristics | (n = 87) | (n = 87) | |
| No. of cycles obtained mature follicles | 83/87 (95.4%) | 84/87 (96.6%) | 0.700 |
| No. of cycles obtained one mature follicle | 59/87 (67.8%) | 40/87 (46.0%) | 0.004 |
| No. of follicles ≥ 18 mm | 1.48 ± 0.92 | 1.68 ± 0.81 | 0.142 |
| No. of follicles ≥ 12 mm | 2.92 ± 1.76 | 3.21 ± 2.35 | 0.371 |
| Progesterone level on hCG injection day, nmol/L | 0.50 ± 1.80 | 0.52 ± 1.57 | 0.873 |
| Estradiol level on hCG injection day, pmol/L | 347.98 ± 300.18 | 283.32 ± 175.14 | 0.095 |
| LH level on hCG injection day, IU/L | 11.13 ± 9.15 | 11.28 ± 13.48 | 0.937 |
| Endometrial thickness on hCG injection day, mm | 9.12 ± 2.25 | 8.57 ± 2.47 | 0.136 |
| Types of endometrial pattern on hCG injection day | B (88.5%) | B (69.0%) | 0.003 |
| The dosage of gonadotropins, IU | 225 [225,375] | 225 [225–375] | 0.274a |
| Characteristics among those obtained mature follicles | (n = 83) | (n = 84) | |
| Endometrial thickness on hCG injection day, mm | 9.17 ± 2.23 | 8.57 ± 2.47 | 0.105 |
| Types of endometrial tissues on hCG injection day | B (90.4%) | B (73.2%) | 0.004 |
| No. of cycles obtained one mature follicle | 59/83 (71.1%) | 40/84 (47.6%) | 0.002 |
| No. of follicles ≥ 18 mm | 1.48 ± 0.92 | 1.68 ± 0.81 | 0.142 |
| Progesterone level on hCG injection day, nmol/L | 0.52 ± 1.57 | 0.48 ± 2.00 | 0.873 |
| Estradiol level on hCG injection day, pmol/L | 347.98 ± 300.18 | 283.32 ± 175.14 | 0.095 |
| LH level on hCG injection day, IU/L | 11.13 ± 9.15 | 11.28 ± 13.48 | 0.937 |
| The dosage of gonadotropins, IU | 225 [225,375] | 225 [225–375] | 0.281a |
| Ovulation | 74/83 (89.2%) | 74/84 (88.1%) | 0.829 |
| Characteristics among those who ovulated | (n = 74) | (n = 74) | |
| Endometrial thickness on hCG injection day, mm | 9.0 [7.0,11.0] | 9.0 [7.0,11.0] | 0.117a |
| Types of endometrial pattern on hCG injection day | B (89.19%) | B (78.38%) | 0.074 |
| No. of follicles ≥ 18 mm | 1 [1, 2] | 1 [1, 2] | 0.003a |
| No. of cycles obtained one mature follicle | 53 (71.6%) | 33 (44.6%) | 0.001 |
| No. of cycles obtained multiple mature follicles | 21 (28.4%) | 41 (55.4%) | 0.001 |
| Progesterone level on hCG injection day, nmol/L | 0.20 [0.09,0.34] | 0.16 [0.05,0.27] | 0.236a |
| Estradiol level on hCG injection day, pmol/L | 268.60 [167.75,429.50] | 246.70 [168.78,381.90] | 0.338a |
| LH level on hCG injection day, IU/L | 9.02 [7.08,10.91] | 8.02 [6.35,10.80] | 0.162a |
| The dosage of gonadotropins, IU | 225 [225,375] | 225 [225–375] | 0.241a |
Values are presented as mean ± SD, n (%) or median (interquartile range). aMann–Whitney U-test
Adverse events
In terms of each type of adverse event, there were no statistically significant differences between the two groups (Table 5). Adverse events occurred in 43 participants in the LE (2.5 mg) + HMG group and 45 in the LE (5.0 mg) + HMG group, and all adverse events were deemed acceptable by the participants. There were three mild OHSS in the LE (5.0 mg) + HMG group, all of which were early-onset OHSS in women who were not pregnant. Two ectopic gestations occurred in the LE (5.0 mg) + HMG group. The LE (2.5 mg) + HMG group included four multiple (twin) pregnancy participants, whereas the LE (5.0 mg) + HMG group had five multiple (twin) pregnancy participants. In the LE (2.5 mg) + HMG group, two NT measurements were abnormal, with one NT value measured at 2.6 mm and the other measured at 2.8 mm, both of which exceeded the normal range, and all of them were isolated with increased nuchal translucency (NT) without any additional abnormalities in the ultrasound. The most common minor adverse events in the two groups included headaches, hot flashes, dizziness, and fatigue.
Table 5.
Adverse events
| Events | 2.5 mg LE + hMG | 5.0 mg LE + hMG | P-value |
|---|---|---|---|
| Side-effects acceptable | 0.762 | ||
| Yes | 43/87 (49.4%) | 45/87 (51.7%) | |
| No | 0/87 (0.0%) | 0/87 (0.0%) | |
| No side-effects | 44/87 (50.6%) | 43/87 (48.3%) | |
| Ovarian hyperstimulation syndrome | 0/87 (0.0%) | 3/87 (3.4%) | 0.246a |
| Early onset OHSS | 0/87 (0.0%) | 3/87 (3.4%) | |
| Late onset OHSS | 0/87 (0.0%) | 0/87 (0.0%) | |
| Other adverse events | |||
| Adverse events before conception | |||
| Hot flashes | 22/87 (25.3%) | 20/87 (23.0%) | 0.723 |
| Headache | 16/87 (18.4%%) | 16/87 (18.4%) | 1.000 |
| Dizziness | 11/87 (12.6%) | 13/87 (14.9%) | 0.660 |
| Abdominal pain | 5/87 (5.7%) | 5/87 (5.7%) | 1.000a |
| Abdominal bloating | 5/87 (5.7%) | 8/87 (9.2%) | 0.566a |
| Constipation | 2/87 (2.3%) | 4/87 (4.6%) | 0.682a |
| Diarrhea | 2/87 (2.3%) | 3/87 (3.4%) | 1.000a |
| Nausea | 6/87 (6.9%) | 9/87 (10.3%) | 0.593a |
| Breast discomfort | 8/87 (9.2%) | 5/87 (5.7%) | 0.566a |
| Joint pain | 5/87 (5.7%) | 4/87 (4.6%) | 1.000a |
| Fatigue | 13/87 (14.9%) | 15/87 (17.2%) | 0.680 |
| Adverse events after conception | |||
| Pregnancy loss | 6/36 (16.7%) | 6/28 (21.4%) | 0.750a |
| Multiple pregnancy | 4/36 (11.1%) | 5/28 (17.9%) | 0.488a |
| Abnormal NT examination of fetus | 2/36 (5.6%) | 0/28 (0.0%) | 0.500a |
aFisher’s exact test
Discussion
This study aims to compare the efficacy of 2.5 mg letrozole and 5.0 mg letrozole in combination with gonadotropins for ovulation induction in infertile patients with polycystic ovary syndrome. The data that we present in this study support a conclusion that LE (5.0 mg) + HMG therapy did not produce a greater benefit than LE (2.5 mg) + HMG therapy in stimulating ovulation and promoting pregnancy, and that there was no significant difference in drug side-effects in this RCT of infertile women with PCOS.
Clomiphene, LE, and HMG are now the mainstream drugs for ovulation induction. Previous studies have confirmed that the efficacy of LE is not inferior [21] or even superior to [22, 23] clomiphene citrate for the treatment of infertility women with PCOS, and there is no significant difference in the incidence of adverse reactions [24]. The most common second-line treatment in infertile women with PCOS is ovulation induction with gonadotropins [25]. It has previously been shown that [26, 27]the incidence of OHSS and other adverse reactions of LE in the treatment of ovulation is significantly lower than that of gonadotrophins monotherapy, and LE therapy makes it easier to obtain one mature follicle, lowering the risk of multiple pregnancies.
According to existing studies [11, 12], the combination of LE and HMG is more effective than LE alone in inducing ovulation and promoting pregnancy in infertile women with PCOS [28]. To probe a better clinical ovulation induction protocol, we examined the therapeutic efficacy of different doses of LE (2.5 vs 5.0 mg) combined sequentially with HMG in the treatment of infertile women with PCOS in the current study. According to the guidelines of the American College of Obstetricians and Gynecologists (ACOG) [29]and contemporary expert consensus statements [25], the current recommendation for infertile patients with PCOS is as follows. The starting dosage is 2.5 mg/day for 5 days following a spontaneous menses or progestin-induced bleed. If ovulation does not occur, the dosage can be increased to 5 mg/day for 5 days with a maximum dosage of 7.5 mg/day. However, recent research is unclear on the best therapeutic dosage of LE in ovulation.
Some studies indicated that LE at 5 mg/day was more effective than 2.5 mg/day for infertile women. The findings were not limited to PCOS patients [14, 30]. A study showed that infertile women (not only PCOS patients) did not benefit more from both 7.5 mg/day of LE and 5.0 mg/day of LE [31], while another RCT study showed that 7.5 mg/day of LE is not as effective as 5.0 mg/day for ovulation induction in women with PCOS [15]. The studies that compared the efficacy of different LE dosages were all plagued by small sample size issues. At the moment, there is a shortage of high-quality clinical studies on the therapeutic impact of different doses of LE used sequentially with HMG in infertile women with PCOS.
In the present study, there was no discernible difference in the ovulation (85.1 vs. 85.1%) or ongoing pregnancy rates (25.3 vs. 33.3%) between the 2.5 mg LE + HMG and group 5.0 mg LE + HMG. Simultaneously, the percentage of type B endometrial pattern on the day of HCG injection was higher in the sequential LE (2.5 mg) + HMG group than in the LE (5.0 mg) + HMG group (P = 0.003). This indicates that sequential LE (2.5 mg) + HMG therapy may be beneficial for improving endometrial receptivity in infertile women with PCOS, but it does not improve the pregnancy rate.
There was no statistically significant difference between two groups in mature follicles rate (P = 0.700); however, the monofollicular development rate was higher in the LE (2.5 mg) + HMG group (P = 0.004). There were no statistically significant differences in multiple pregnancies or incidence of OHSS between the two groups. Our results suggest that LE (2.5 mg) + HMG is more beneficial for patients with PCOS in promoting single follicular development.
We also performed a stratified analysis by BMI, insulin resistance, and WHR to probe the factors affecting ovulation and ongoing pregnancy rates. The ovulation and ongoing pregnancy rates were not significantly different in any stratum between the two groups. Based on the stratified analysis results, BMI, WHR, and insulin resistance could not predict the outcomes. The outcome revealed a positive effect or tendency for ongoing pregnancy following sequential LE (2.5 mg) + HMG treatment in each stratum; however, the difference was not statistically significant. This may be because of the small sample size. Additional clinical studies with larger sample sizes are needed to confirm these findings.
Previous studies have shown that higher doses of LE have an adverse effect on the endometrium of infertile women (not only patients with PCOS) who undergo ovulation induction treatment [15, 31]. Furthermore, LE at 5 mg/day or 7.5 mg/day may result in multi-follicle development compared to LE at 2.5 mg/day. Therefore, a lower dose of LE (2.5 mg/day) may improve endometrial receptivity when used in conjunction with HMG. Moreover, LE (2.5 mg) + HMG was more beneficial in promoting single follicular development in patients with PCOS. Furthermore, LE (5.0 mg) + HMG treatment resulted in more mature follicles, increasing the risk of OHSS and multiple gestations.
One of the innovative points of the present study was that the participants were infertile women with PCOS, which differed from those of previous studies on the therapeutic efficacy of different doses of LE. Another innovative aspect of our study was that we probed the treatment efficacy of different doses of LE in HMG sequential therapy, which has not been investigated in previous studies. Our results show that patients in the 5.0 mg LE group produced more mature follicles than those in the 2.5 mg LE group when combined with HMG, which was similar to the results of previous studies [15, 31]. Additionally, patients in the LE (2.5 mg) + HMG group may have better endometrial receptivity than those in the LE (5.0 mg) + HMG group. In previous studies [14, 30], LE monotherapy (5.0 mg) was superior to LE monotherapy (2.5 mg) in infertile women (not only patients with PCOS). However, according to the results of our study, when infertile women with PCOS were treated with a higher dose of LE (5.0 mg/day) during induction of ovulation with LE combined sequentially with HMG therapy, they did not experience better therapeutic efficacy but may increase the risk of OHSS and multiple pregnancies.
Based on the stratified analysis and cycle characteristics, LE (2.5 mg) + HMG may be an optimal treatment dose when doctors perform sequential combination treatment of HMG. We performed a detailed investigation of the sequential combination regimen and provided novel evidence to establish a basis for the selection of treatment protocols for ovulation induction in infertile women with PCOS. Further studies are required to explore the effects of LE on endometrial receptivity and its mechanisms of action.
Insulin resistance is common in PCOS patients. It is related to the absence of ovulation (anovulation) and infertility [32]. There was no difference in fasting glucose, fasting insulin, and HOMA-IR between the two groups. Therefore, it remains unclear whether different doses of LE combined with HMG are effective for ovulation promotion in PCOS patients with insulin resistance. Meanwhile, our diagnostic criteria are based on the generic PCOS diagnostic criteria, which may be used as a reference in other clinical settings to treat the same population. In this article, we analyzed not only outcome measures for all participants but also cycle outcome measures and cycle characteristic indicators, which can intuitively highlight the differences between the two groups. It has the potential to offer a scientific foundation for clinical trials.
One of the limitations of our study is the modest sample size, with only 174 participants enrolled. This restricted number of participants may affect the ability to generalize our findings to a broader population. Consequently, we recommend that future studies aim to include a more substantial sample size to enhance the external validity of the findings. Another limitation of this study is that, as shown in Tables 1 and 3, the number of subjects with overweight and obesity among those included in the study is relatively small. Considering that most clinicians would encounter a significant number of overweight and obese women with PCOS in their practice, this represents another limitation of the study in terms of generalizing these results across women of all BMI ranges. Therefore, ovulation induction treatment for overweight and obese PCOS patients may require further clinical research focusing on obese PCOS patients in the future to provide more robust evidence. An additional point that warrants discussion is that the inclusion and exclusion criteria of this study did not take into account whether patients had a history of tuberculosis. Since pelvic tuberculosis can lead to adhesions in the fallopian tubes, which in turn can increase the incidence of ectopic pregnancy, it is recommended that future studies should further refine their research design to address this issue. Specifically, during the process of enrolling and excluding subjects, patients with patency in only one fallopian tube should be screened to exclude those who may have pelvic tuberculosis infection (for example, through endometrial biopsy, tuberculin skin test, or blood tests for tuberculosis).
Conclusion
In summary, the present study demonstrate that LE (2.5 mg/day) combined with HMG may be an optimal treatment dose that can improve endometrial receptivity and promote single-follicle development in PCOS patients. A higher dosage of LE (5.0 mg/day) followed by HMG treatment showed a higher multifollicular development rate. It may increase the risk of OHSS and multiple pregnancies in this group.
Acknowledgements
We thank all study participants and all members of the Reproduction Centre of Wuhan Union Hospital for their assistance in patient recruitment.
Author contribution
YL contributed to the experimental design, project development, data collection, and manuscript editing. JL conducted manuscript writing and participated in data collection and data analysis, ethical application, and registration application for the clinical trial. WX participated in experimental design, data collection, and management. YP participated in data collection and data analysis. XD participated in data collection. XW participated in project development. WL participated in project development. XL participated in data analysis. LZ participated in data collection. YF participated in data collection and manuscript editing. All authors have reviewed the manuscript and approved the final version.
Funding
This work was supported by grants from the Natural Science Foundation of Hubei Province, China (No. 2023AFB506).
Availability of data and materials
The datasets generated and/or analyzed during the current study are not publicly available due to the sensitivity of the data but are available from the corresponding author on reasonable request.
Declarations
Ethical approval
The study was approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, and the approval number was 0121–01. All participants had informed consent.
Consent for publication
All authors consent to the publication of this work and authorize granting this license.
Conflict of interest
The authors declare no competing interests.
Footnotes
Jingyi Li and Yuan Peng are co-first authors, Jingyi Li is the primary first author.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Contributor Information
Wenqian Xiong, Email: 176051948@qq.com.
Yi Liu, Email: liqun1994@hust.edu.cn.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets generated and/or analyzed during the current study are not publicly available due to the sensitivity of the data but are available from the corresponding author on reasonable request.