In vitro fertilization (IVF) requires controlled ovarian stimulation to drive multiple follicle development and to optimize the number of retrieved oocytes. The regimen for controlled ovarian stimulation typically employs gonadotropins such as recombinant follicle-stimulating hormone (FSH), including follitropin alpha or follitropin beta, or human menopausal gonadotropin (hMG) [1, 2, 3]. Follitropin delta is a contemporary recombinant FSH analogue synthesized in a human cell line and can be administered at a dose calculated based on an algorithm that considers the anti-Müllerian hormone (AMH) level and body weight of each individual [4]. This is regarded as a step toward a more individualized ovarian stimulation in IVF. In recent years, numerous randomized controlled trials (RCTs) have assessed the clinical efficacy and safety of follitropin delta in IVF and have compared these findings with those for traditional gonadotropins [5, 6, 7]. The evidence from these investigations indicates that the individualized dosing strategy of follitropin delta can yield a comparable IVF success rate to conventional gonadotropin protocols [5, 6, 7, 8]. Clinical outcomes from an IVF cycle, including clinical pregnancy, ongoing pregnancy, and live birth rates, are statistically comparable to those achieved with fixed-dose follitropin alpha/beta or urinary gonadotropin [5, 6]. While some studies demonstrated that follitropin delta can yield a tendency for improved outcomes [5, 6, 7], a comprehensive meta-analysis revealed no significant difference in overall pregnancy outcomes [8]. Therefore, in terms of clinical IVF practice, safety outcomes, e.g., ovarian hyperstimulation syndrome (OHSS), must be investigated in addition to the effectiveness before any new ovarian stimulation protocols are adopted. Recent RCTs consistently demonstrate a significantly reduced incidence of OHSS or excessive ovarian response following follitropin delta use compared to regular gonadotropins [5, 6, 8, 9]. The finding regarding the improvement in safety outcomes associated with follitropin delta observed in the initial cycle may be extrapolated to subsequent treatment cycles [9]. This can be a therapeutically important benefit, as OHSS may result in hospitalization and the cessation of the cycle. Because the dose of follitropin delta is calculated based on serum AMH and body weight, it is necessary to validate the clinical effectiveness of follitropin delta across diverse groups of women based on these biomarkers. Alternatively, clomiphene citrate (CC) and letrozole have both been used with gonadotropins in poor responders and have been shown to reduce the amount of gonadotropin used without reducing the pregnancy rate [10].

Prior studies have directly compared follitropin delta with traditional gonadotropins as two separate arms and measured the outcomes in both groups. This parallel study design may introduce confounders due to inter-patient variability, including variations in age, ovarian reserve, and underlying fertility diagnoses. The clinical interpretation of this design illustrates how clinicians should select the type of gonadotropin in the first cycle, taking into account baseline characteristics. However, we argue that a pre–post design, wherein women are administered conventional follitropin (e.g., alpha or beta) in the first IVF cycle, followed by follitropin delta in the subsequent cycle, provides a substantial benefit by utilizing each participant as the control. This within-subject comparison may help mitigate the impact of differences in baseline characteristics and the potential for post-randomization bias [11], facilitating decision-making in the context of ovarian stimulation after a failed IVF cycle. Therefore, this study aimed to assess embryological outcomes using a pre-post design to evaluate whether the combination of follitropin delta and CC offers advantages over follitropin alpha in women with a low prognosis.

This study evaluated the embryological outcomes of IVF cycles stimulated with the combination of follitropin delta and CC in a cohort of women who previously had suboptimal responses to stimulation with follitropin alpha. Using a pre-post study design, we observed an improvement in embryology outcomes, including a significant increase in the number of day 3 embryos, day 5–6 embryos, and good-quality blastocysts from follitropin delta plus CC cycles compared to follitropin alpha cycles. These findings suggest that follitropin delta may offer advantages in embryology quality over conventional gonadotropins, particularly in patients who have previously demonstrated suboptimal responses to ovarian stimulation.

The study results are consistent with previous findings, which indicate that follitropin delta produced superior embryological outcomes compared to traditional gonadotropins. Prior RCTs documented that follitropin delta provided comparable pregnancy rates to follitropin alpha while reducing the occurrence of OHSS through its tailored dosing approach [9, 23].

The findings of this study differed from certain other meta-analyses, indicating no substantial difference in blastocyst quantities between follitropin delta and conventional gonadotropins [8]. Therefore, these improvements in embryological outcomes may be attributed to the pre-post design of this study, wherein each participant acted as their own control, thereby reducing interpatient variability. Moreover, our cohort comprised women who had previously shown a suboptimal response to follitropin alpha, indicating that follitropin delta may be particularly beneficial for this subgroup.

A plausible explanation for the increase in the number of high-quality blastocysts in our study is the enhanced follicular recruitment and synchronization noted with follitropin delta plus CC. Prior pharmacokinetic investigations have shown that follitropin delta demonstrated reduced FSH clearance and prolonged action compared to recombinant FSH sourced from Chinese hamster ovary cells [4]. This pharmacodynamic profile may have resulted in more uniform follicles.

The pre-post design used in this study represents a major strength since this design facilitated a direct comparison of ovarian stimulation regimens within the same woman. This design clarified the impact of alterations in gonadotropin regimen on embryological outcomes by removing interpatient variability. To our knowledge, this was the first pre-post study to evaluate the effectiveness of follitropin delta, combined with CC, in patients who had previously responded suboptimally to follitropin alpha.

This research has some limitations. The primary limitation of this study is its modest sample size. A post hoc power analysis suggests that this study was underpowered, particularly for detecting small to moderate effects (data not shown). Consequently, while we observed a statistically significant increase in the number of high-quality embryos, the precise magnitude of this effect should be interpreted with caution, as it may represent an overestimation of the actual impact. Our analysis involved assessing seven related embryological outcomes. While a strict statistical correction (e.g., Bonferroni) was not applied due to the highly correlated nature of these endpoints within a single biological pathway, we acknowledge that this approach carries a potential risk of an inflated Type I error. Therefore, our findings should be considered exploratory and hypothesis-generating, underscoring the urgent need for larger, prospective randomized trials to confirm these promising results and provide a more precise estimate of the treatment effect.

Another limitation of our study is the retrospective pre-post design. Although the mixed-effects models in our research minimize interpatient variability, it is susceptible to a potential cycle sequence effect from the initial stimulation cycle. Although we managed to adjust for this effect by adding the time interval between cycles as a covariate, we cannot completely exclude the possibility that prior ovarian stimulation influenced the outcomes of the second cycle.

The lack of an external control group was also a limitation of the study. A comparative analysis with women who received follitropin alpha in their second cycle would yield a more thorough evaluation of the relative efficacy of follitropin delta combined with CC. A parallel design was necessary to compare outcomes as study participants transitioned from follitropin delta cycles to follitropin alpha cycles. Nevertheless, our within-subject comparison mitigated numerous confounding variables that commonly influence parallel-group research, including baseline ovarian reserve and individual heterogeneity in response to gonadotropins.

This study was limited in that it focused solely on embryological outcomes and did not provide evidence on pregnancy outcomes, including live birth rates. Although improved embryo quality was a beneficial aspect, it remained uncertain if these improvements could be translated into improved pregnancy outcomes. Hence, further research investigating implantation rates, clinical pregnancy rates, ongoing pregnancy rates, and live birth rates [24, 25] is essential to ascertain the comprehensive clinical effects of follitropin delta plus CC.

One of the statistical issues with modeling count outcomes is the presence of zero-inflation and overdispersion. To confirm the robustness of this key finding on the day 5 good-quality embryo outcome, a sensitivity analysis using a zero-inflated negative binomial (ZINB) model was performed. This model yielded a result (IRR: 2.00; 95% CI 1.11–3.59), which was highly consistent with that of the ZIP model. Therefore, the more parsimonious ZIP model was retained for the final analysis based on its lower AIC. Our study investigated seven downstream embryological outcomes of an IVF cycle. A strict statistical correction (e.g., Bonferroni) was not applied, as formal correlation analysis confirmed that the seven outcomes are highly interdependent (e.g., Spearman’s r $>$0.80 between sequential endpoints), violating the assumption of independence required for such corrections.

The findings of this study have numerous clinical implications. Indeed, a key finding of this study was that the second cycle, combining follitropin delta and CC, resulted in a statistically significant increase in the number of total day 3 embryos and, most notably, a nearly two-fold increase in the rate of good-quality day 5–6 embryos (IRR: 1.90). While this did not stem from a significantly larger pool of retrieved oocytes, the downstream improvement in embryo yield is of high clinical relevance. The value of each additional embryo, particularly at the blastocyst stage, is well-established. Large-scale data show a direct correlation between the number of usable blastocysts and the cumulative live birth rate, with each additional blastocyst providing a patient with an extra, distinct opportunity for transfer and conception [26]. Thus, the enhanced ability to generate high-quality blastocysts, as observed in our study, represents a clinically significant outcome that can potentially improve the overall chance of success for each patient from a single stimulation cycle. Another important clinical consideration is the potential safety benefits of follitropin delta combined with CC. Previous studies have reported a lower risk of OHSS with follitropin delta compared to conventional gonadotropins [9]. Although our study did not specifically assess OHSS incidence, the use of follitropin delta, which allows for individualized dosing regimens, may contribute to a safer approach to ovarian stimulation [9].

In summary, this study provides novel insights into the use of a combination of the follitropin delta and CC in women undergoing IVF with a prior suboptimal response to follitropin alpha. The findings suggest that follitropin delta combined with CC may result in better embryo quality, potentially improving IVF success rates in this specific population.

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Conception and design: HDT, STT and TNT; Administrative support: DKN; Provision of study materials or patients: TNT and STT; Collection and assembly of data: HDT, NNN, TTV and PNM; Data analysis and interpretation: HTMP, TQV, TVT, HHS and DKN. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All participants had provided written informed consent of their clinical data to be used for research purposes, in accordance with the ethical principles delineated in the Declaration of Helsinki. This study received approval from the Institutional Ethics Committee of the Vietnam Military Medical University under approval number 01/2024/CNChT-HĐĐĐ dated August 6, 2024.

This research received no external funding.

The authors declare no conflict of interest.

During the preparation of this work, the authors used ChatGPT in order to check spelling and grammar. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.