The present study is based on a broad population of patients undergoing COS and IVF/ICSI with fresh embryo transfer and validated with a reliable and rigorous analysis revealing that repeated COS-IVF/ICSI does not impact ovarian reserve function; however, it influences the clinical pregnancy outcome. We ascertained that the population with a higher number of repetitions ended up with the worse clinical pregnancy outcome. Intriguingly, there exhibited opposite results in the population with the same number of repetition that multiple COS-IVF/ICSI enhanced CPR in Groups B, C, and D and ameliorated LBR and EMR in Groups B and C. However, the modifications in CPR, LBR, and EMR did not reach the statistical significance up to 5 repetitions, implying that COS-IVF/ICSI in the cohort of repeated cycles ≤ 4 times in infertile patients is favorable for pregnancy outcomes. The present study is noteworthy in that, to the best of our knowledge, it is the most extensive study to date examining the effects of repeated COS on ovarian reserve and clinical pregnancy outcomes in fresh cycles.
Ovarian reserve, which symbolizes the number of oocytes retained in the ovary, is more influenced by factors such as genetics, age, and environment [8]. Hence a population with a repertoire of multiple COS was chosen for self-control comparison to amplify the reliability of the study findings. The current study guidelines tailored AMH and AFC as the most representative predictors of ovarian function, the simplest, the most sensitive, and superior to day-3 FSH [8, 9]. Other indicators can be used as auxiliary judgments. The AMH, AFC, basal FSH, and basal LH levels were not significantly different among cycles in Groups B, C, D, and E, whereas only basal E2 levels aggrandized with the increase in cycle number. The present data suggested basal E2 levels as the only suitable auxiliary indicator for precisely interpreting normal basal FSH values and hampered its use as an ovarian reserve function indicator [9]. Therefore, findings revealed that repeated COS does not affect ovarian reserve function.
A systematic review by Luk et al [1] concluded that repeated COS does not negatively impact the ovarian reserve function within ≤ 3 cycles. Xu et al [10] picked basal FSH, LH, E2 levels, and AFC to assess ovarian reserve function and demonstrated that the impact of COS on ovarian reserve function was not significant up to 4 cycles, but they did not compare different groups for the difference in AMH levels between cycles. This study further indicates that 5 treatment cycles still do not affect ovarian reserve function. In contrast, the puncture during the retrieval of oocytes damages the capillaries and follicles of the ovary [1]. A previous study has observed that high concentrations of anti-ovarian antibodies in infertile patients undergoing multiple IVF caused by repeated punctures were correlated with a decrease in eggs after stimulation [11]. Whether COS after 5 repeats negatively correlates with ovarian reserve function is, therefore, necessary to be investigated.
The present study revealed that the clinical pregnancy outcomes measured by the CPR, LBR, and EMR deteriorated considerably as repeated populations increased. The findings of the regression analysis revealed that decreasing age (aOR = 0.96; 95% CI, 0.95–0.97; P = 0.000) and increasing AFC (aOR = 1.02; 95% CI, 1.01–1.03; P = 0.000) significantly influenced clinical pregnancies, which implied increasing age and declining ovarian reserve function are potential reasons for performing COS-IVF/ICSI repeat. Poor clinical pregnancy outcomes associated with older age and more repeat cohorts are apprehensible, yet the number of repeats still significantly affects clinical pregnancy outcomes after controlling potential confounders such as age, BMI, and AFC. Furthermore, the CPR in the cohort ≤ 4-cycle and the LBR in the ≤ 3-cycle cohorts were found significantly higher after multiple COS-IVF/ICSI by self-control analysis in the same repeat count population. The findings revealed that repeat COS in ≤ 4 cycles positively affects clinical pregnancy outcomes while the CPR at Cycle 5 is insignificant to the clinical pregnancy outcomes.
Homburg et al. [12] reported a considerable decrease in CPR after three cycles, but this study did not perform a further regression analysis to obtain more reliable results. Likewise, another study strengthened that repeated COS did not negatively affect CPR and LBR in patients experiencing three cycles and less until after > 3 cycles [1]. A multicenter study by Meldrum et al. [13] illustrated a significant reduction in CPR for > 4 cycles and LBR for ≥ 4 cycles. It is reassuring that the present study came up with the similar results. Additionally, there was a significant enhancement in oocyte number, fertilization outcome, and embryo quality upon increasing the number of cycles beyond 3 COS repeats in egg donors [14]. Similarly, another study reported a substantial increase in fertilization and CPR in patients with repeated COS cycles compared to unrepeated cycles saving did not further differentiate repeated cycles by number [6]. Nevertheless, the CPR was stable in patients with 4 to 8 cycles under 42 years of age, hence defining the appropriate age and cycles for patients after analyzing pregnancy outcomes from 4 (169 women) to 8 (27 women) cycles of treatment [15].
The cumulative effect of three complete IVF/ICSI cycles (hereafter referred to as multiple cycles) is considered by NICE to increase the chance of successful pregnancy to 45–53% in infertile patients under 40 years of age, which is the most cost-effective and clinically efficient option [16]. Thereby, the organization proffers that IVF/ICSI treatment should be planned on a multi-cycle basis. The present study stated that the CPR of three and four consecutive complete cycles reached 43.44% and 52.50%, respectively. A cross-sectional survey conducted by Harrison et al. [17] in 2021 on willingness to plan for multiple cycles contended that most patients preferred three completed cycles of IVF/ICSI (referred to as multiple cycles) treatment, unlike the current cycle-by-cycle treatment plan. In addition, emotional and physical stress are the main factors attributable to many infertility patients who discontinue treatment rather than financial costs [18]. Therefore, the patients undergoing treatment or experiencing treatment failure needed expert counseling and encouragement to enlighten the spark of hope among them. If the ART treatment process is defined as a multi-cycle process, pre-planning the multi-cycle treatment is necessary to make fully aware of the patient with associative risk of implantation and pregnancy failure and discuss the treatment strategy to follow after treatment failure with patients in advance in order to achieve patients' parenting goals sooner and better [17, 19]. More research is needed in the future to evaluate the feasibility and acceptability of this treatment planning approach.
This study exhibited that a raise in cycle number significantly increases CPR in the cohort of ≤ 4 repeated cycles, improves LBR, and reduces EMR in cohorts with ≤ 3 repeated cycles. The validity of advising patients to actively try 4 attempts in this study can be derived from the above study results, which enlighten hope and encouragement in infertile patients. Consequently, the patients are not advised for another attempt; however, adoption may be considered a feasible option if infertile patients do not have a successful pregnancy after 4 attempts based on cost and pregnancy outcome considerations. Nevertheless, the possibility of achieving clinical pregnancy with normal birth after 4 cycles is not denied.
The small number of patients undergoing COS > 5 times in the clinic makes statistical analysis difficult, whereas a distinctive study can be designed to analyze them in an animal model. Antonouli et al. [20] reported that the oviducts of mice receiving 4 and 8 gonadotropin stimulation did not perceive significant morphological changes but underwent significant ultrastructural alterations, including a dramatic decrease in ciliated cells and massive mitochondrial degeneration compared to controls (who did not receive stimulation). These alterations were more pronounced at 8 than 4 times, which may negatively affect the fertilization process and embryo transport to the uterus. The research team working on this topic previously demonstrated that repeated (6 and 8 times) gonadotropin stimulation leads to overexpression or overactivation of intracellular localization/content of proteins controlling cell cycle progression in the oviduct and ovary of mice, causes damage to the oocyte spindle, which reduces reproductive performance in mice [21]. Nevertheless has no significant effect on mice that suffer 4 times stimulation [22].
Likewise, the first stimulation cycle resulted in a 3-fold increase in oocytes, yet the number of oocytes decreased as the number of stimulations increased. The frequency of mitochondria in mouse oocytes with uneven distribution increased significantly, increasing spindle breakage and intracellular oxidative stress and decreasing expression of octamer-binding transcription factor 4 (Oct4) after four cycles [23]. Nie et al [24] created a mouse model of premature ovarian failure (POF) by more than 10 successive COS. They detected that successive COS induced ovarian oxidative stress, apoptosis, and ovarian damage through various mechanisms, including p16 and SIRT1/FOXO1 signaling pathways, reduced ovarian function and oocyte quality. However, it is manifested that repeated ovarian stimulation (10 times) significantly declined the number of primary follicles compared to single ovarian stimulation. In contrast, it did not affect autophagy, proliferation, apoptosis, and aging of the ovary [25].
These mice trials reported the detrimental effects of recurrent ovarian stimulation on the integrity of the fallopian tubes, ovaries, and oocytes and the potential to increase the risk of gynecological malignancies. Clinical studies have also revealed that COS-induced physiologically high levels of E2 may increase the risk of cancer in estrogen-sensitive tissues such as ovarian, uterine, and breast tissues [26–28]. Yet, the issue remains controversial, with some researchers’ findings denying an increased risk of cancer in the COS group, possibly due to inconsistent drug exposure times and short follow-up periods [29, 30]. In addition, it has been demonstrated that repeated COS increases the venture of osteoporosis and cardiovascular disease by accelerating ovarian aging [31].
Although the mouse model is the most commonly used model for clinical studies, the present mouse experimental evaluations yielded many exciting results. What cannot be ignored is the variability between humans and mice, and the primate model is perhaps more accurate. Dong et al [32] revealed that although no significant abnormalities in follicle activation and growth and no serious damage to ovarian structure and function in repeatedly receiving COH in rhesus monkeys after 5 years. Nevertheless, granulosa cells of Mitochondria were impaired, in which StAR and P450arom protein expression was downregulated, inhibiting cell proliferation and differentiation and possibly negatively impacting ovarian function. The team also explored the effects on uterine and mammary tissues after 5 years in rhesus monkeys experiencing four COS. It proved that uterine cells displayed a low proliferative state and mammary epithelial cells depicted low proliferation and high apoptosis, and no cancer risk was observed in both [33]. However, the small sample size limited both studies and their conclusions (experimental group n = 3). Larger sample sizes and longer-term studies would provide a more comprehensive picture of the question under investigation.
The potential adverse effects of repeated COS on ovarian function were detected in both the mouse model and the rhesus monkey model above, yet did not produce serious damage to their phenotypes, which abnegate the adverse effects of repeated COS on ovarian reserve function in clinical case-control studies. Nevertheless, the possibility of significant functional changes in these organs after a longer period of potential damage cannot be guaranteed.
The strength of this study is the utility of self-control comparisons since it diminishes the influence of numerous uncontrollable factors such as genetics and environment, making the results and conclusions more reliable. Second, the study used univariate comparisons and multifactorial logistic regression analyses to compensate for potential confounding factors' effects on clinical pregnancy outcomes. Third, strict exclusion criteria were implemented to preclude patients with PCOS, endometriosis, and genetic factors for infertility and patients with ovarian hyporesponsiveness with FSH > 15 U/L and AFC < 3. Fourth, the study explored the effect of repeat COS on clinical pregnancy outcomes, an aspect less frequently investigated in articles, and scant information are available in publications. Moreover, to our knowledge, this is the largest sample size, and the subgroups are more detailed, making the findings more convincing.
A drawback of the present analysis is that it is an observational study with unavoidable inherent flaws and is unable to collect and control for all possible confounding factors. Still, the study used a reliable study design. The self-control comparison itself has reduced the uncontrollability of retrospective studies. Additionally, the data from this study only allowed grouping to the 5-repeat cycle population, and exploration was not extended to the most frequent populations. However, the effect of the number of repeats on clinical pregnancy outcomes has reached an inflection point, where CPR no longer increases significantly after 4 times.