Effect of follicle size on pregnancy outcomes in patients undergoing first letrozole-intrauterine insemination

Intrauterine artificial insemination (IUI) after ovulation induction is often used for the treatment for infertile couples with anovulation, mild male factor infertility, endometriosis, or unexplained infertility [1, 2]. Many factors have been reported to affect the success of IUI treatment cycles [3‐5]. One of these factors is the size of dominant follicle before triggering.

As we all know, the size of follicular is an indirect marker of oocyte maturity, the timing of human chorionic gonadotropin (hCG) trigger based on follicle size is crucial for the success of IUI cycles [6]. However, there is no general consensus on the ideal size of the follicle in the literature. A variety of studies have investigated the dominant follicle size in IUI cycles using ovulation induction drugs of clomiphene citrate (CC) or gonadotropins, yielding inconsistent findings. For instance, a study of 1483 CC-IUI cycles demonstrated that women with follicular diameters measured 20 mm or greater exhibited lower pregnancy rates compared to those with follicle diameters ranging from 15.00 to 19.99 mm [7]. Conversely, another study observed higher pregnancy rates when the leading follicles measured between 23 and 28 mm [8]. However, there is little clinical research foused on the effect of follicle size on pregnancy outcomes during letrozole-IUIs.

Letrozole, a frequently employed pharmacological agent in clinical settings for promoting ovulation, is classified as a third-generation nonsteroidal aromatase inhibitor. Its mechanism of action involves the reduction of estrogen levels in patients by impeding the conversion of androgens to estrogens, thereby stimulating the hypothalamus-pituitary gland to secrete increased levels of gonadotropins to facilitate ovulation [9]. In the treatment of unexplained infertility, letrozole has been shown to be as effective as clomiphene citrate with a lower risk of multiple births [10]. It is more effective to induce ovulation with letrozole than with clomiphene citrate in patients with polycystic ovary syndrome by using letrozole [11].

In the present study, we performed a retrospective clinical investigation to explore the effect of leading follicle size for hCG trigger on pregnancy outcomes in patients undergoing first letrozole-IUI.

In the present study, we found that in the first letrozole-IUI cycle, the optimal follicle size before hCG trigger was significantly associated with PR, CPR and LBR, even after adjusting for known confounders including female age, BMI, infertility type, the duration of infertility, infertility diagnosis, endometrium thickness, FSH, AMH, TMC, LH surge and E2 on the day of hCG trigger. The optimal follicle size range on the day of hCG trigger was 19.1–21.0 mm, which resulted in the highest PR, CPR and LBR. In addition, we also investigated the impact of estrogen levels on the day of hCG trigger on the outcome of the IUI cycle. When the E₂ level was low than 200 pg/mL, there was a significant difference among the six follicle-size groups in clinical pregnancy rates. However, we did not observe a similar difference when the E₂ level was more than 200 pg/mL.

Several studies have tested the predictive value of dominant follicle size on the success of IUI cycles. One study found that the most favorable lead follicle size in hCG-triggered letrozole-human menopausal gonadotropin IUI cycles ranged from 16.1 to 18.0 mm [14]. However, in another study, the size of the dominant follicle on hCG day in gonadotropin and IUI cycles did not demonstrate a statistically significant impact on pregnancy outcomes [15]. Inconsistent findings regarding the optimal size of lead follicles, ranging from 18 to 22 mm, have been reported in various studies [16‐18]. Ghosh et al. discovered an inverse relationship between follicular diameter and pregnancy outcomes in clomiphene citrate IUI cycles. They observed that women with follicle sizes ranging from 15.00 to 19.99 mm had the highest likelihood of achieving pregnancy [7]. The authors proposed a classification system for follicles, categorizing them as immature (< 15 mm), mature (15–2 mm), and postmature (> 23 mm). Consequently, an excessive number of large follicles (≥ 20 mm) may decrease the chances of conceiving. Our study has drawn conclusions similar to this study, which also suggested either too large or too small follicles may lead to a decrease in pregnancy rate.

Although there is no general consensus on the ideal size of the follicle in the literature, the practice is that HCG is applied when the size is 18 mm or higher. Therefore, our study set < 18 mm as the smallest follicle group. Meanwhile, we also incorporated a greater number of follicle-size categories, aiming to more accurately evaluate pregnancy outcomes across a precise range of follicle sizes. Serial transvaginal ovarian ultrasounds with follicle measurement were performed by a fixed ultrasound physician in our study. The dominant follicle was measured from inner edge to inner edge to the nearest 0.1 mm in 2 perpendicular axes by transvaginal ultrasound. Our results showed the optimal follicle size ranging on the day of hCG trigger was 19.1–21.0 mm. In our study, although 21.1–22.0 mm group was associated with an increase in the likelihood of pregnancy and the clinical pregnancy, but not with live birth. Kolbe et al. also have found that the lead follicle size ranged from 21.1 to 22.0 mm was correlated with increased likelihood of clinical pregnancy among individuals undergoing their initial CC-IUI cycles, but unfortunately they did not follow-up the live birth rate [19]. Two other studies have also confirmed that follicles that are excessively small or excessively large will not produce mature oocytes and will not be suitable for fertilization [20, 21].

Ovulation induction drugs in previous studies were CC, or gonadotropins, CC combined with gonadotropins, or letrozole combined with gonadotropins, there is limited research on IUI for single letrazole. It was as effective as CC in inducing ovulation with letrozole. Rachmawati A et al. found that follicle sizes ranging from 18 to 22 mm in both the CC and Letrozole groups increased biochemical pregnancy rate. Furthermore, the use of Letrozole resulted in a 1.513 times higher biochemical pregnancy rate compared to CC [3]. These findings suggest that follicle sizes within the 18–22 mm range and the utilization of Letrozole as an ovarian stimulator are predictive factors for a higher pregnancy rate in women undergoing IUI.

Letrozole has gained widespread usage in the controlled ovarian hyperstimulation treatment of unexplained infertility and is now the preferred method of ovarian induction in anovulatory women with polycystic ovary syndrome (PCOS) [11, 22]. As a nonsteroidal aromatase inhibitor, letrozole exerts an antiestrogenic systemic effect by inhibiting the production of estradiol. In letrozole cycles, it is common to observe the presence of a mature follicle during the late follicular phase through ultrasound assessment, concurrent with a subphysiologic serum estradiol level, as granulosa cell aromatase activity has not yet rebounded from inhibition [23]. Therefore, we investigated the effect of E₂ levels on the outcome of the IUI cycle on the day of hCG trigger. When we performed stratified analysis by subgrouping patients according to the levels of E₂ on hCG-trigger day, there was no significant difference in pregnancy outcomes between the group with low and higher estradiol concentrations. Interestingly, when the E₂ level was low than 200 pg/mL, there was a significant difference among the six follicle-size groups in pregnancy outcomes. However, when the E₂ level was higher than 200 pg/mL, there was no difference. The potential anti-fertility effects of lower E₂ in the ovulation induction treatment with letrozole, could include effects on the endometrium, periovulatory uterine contractions, fallopian tube function, and oocyte quality [24]. We just guess that high estrogen levels mean granulosa cell aromatase recovered from aromatase inhibition. When the follicle was with high estrogen concentration in letrozole cycle, the leading follicle size on the day of triggering would not affect the success of IUI. Therefore, we can speculate that using follicle size as a predicator of pregnancy outcomes is more meaningful when estrogen on the day of hCG trigger is less than 200 pg/ml. The potential impacts of letrozole with regard to its resulting lower E₂ levels remain to be further studied.

A strength of this study is to only include patients diagnosed with unexplained infertility and anovulation, and to include the first IUI cycle with letrozole alone. This study distinguishes itself from previous research by incorporating a greater number of follicle-size categories, enabling a more precise assessment of pregnancy outcomes across a precise range of follicle sizes. The assessed endpoint of this study was live birth, which provided a more precise measure of the success rate of IUI. The current study is limited by its retrospective study design, which introduces the potential for confounding bias due to unmeasured confounding variables. In addition, the size of the follicles was determined by averaging the diameters of the follicles in 2 perpendicular axes in this study. For this reason, our data may not be applicable for the main follicle sizes measured using different techniques, and the variability of follicle size measurements might be a confounding factor in this study. And also it is a single center study with small sample size, so this population was underrepresented in the results.

In conclusion, the optimal leading follicle size associated with better pregnancy outcomes ranges from 19.1 to 21.0 mm in individuals undergoing their initial letrozole-IUI cycles trigged by hCG, especially when estrogen on the day of hCG trigger is less than 200 pg/ml. According to present results, healthcare providers can determine the timing of HCG trigger based on the follicle size. Nevertheless, it is important to note that this study is retrospective in nature, thus necessitating additional well-designed prospective cohort studies to validate the findings.