Fertility preservation has become an increasingly important part of care for young women diagnosed with breast cancer. As survival continues to improve, more patients are asking not only how to treat the disease, but also how to protect their future reproductive options before starting systemic therapy.
One of the main approaches is oocyte cryopreservation, which requires controlled ovarian stimulation. In breast cancer, especially hormone-sensitive disease, this has raised an important clinical concern: could the temporary hormonal changes caused by stimulation influence tumor biology?
A new prospective paired-biopsy study published in ESMO Open provides reassuring biological data on this question. The study evaluated whether controlled ovarian stimulation with concurrent letrozole produced short-term histological or molecular changes in breast tumor tissue.
A Study Designed to Look Directly at the Tumor
The study included 21 premenopausal women younger than 40 years with breast cancer who were candidates for fertility preservation before neoadjuvant systemic therapy at the Breast Cancer Unit of the Clínic Barcelona Comprehensive Cancer Center.
All patients underwent controlled ovarian stimulation using recombinant follicle-stimulating hormone with concurrent letrozole at 5 mg daily. A random-start protocol was used, allowing stimulation to begin regardless of menstrual-cycle phase and helping avoid unnecessary delays before cancer treatment.
Importantly, the study used paired tumor samples. Researchers compared the original diagnostic biopsy with a second tumor biopsy obtained within 24 to 48 hours after oocyte retrieval. This design allowed investigators to assess whether ovarian stimulation was associated with direct changes in tumor tissue.
The analysis included immunohistochemistry for estrogen receptor, progesterone receptor, HER2, Ki-67, and tumor-infiltrating lymphocytes, as well as PAM50 gene-expression profiling and risk of recurrence scoring.
No Significant Changes in Key Tumor Markers
The main pathological findings remained stable after ovarian stimulation.
No significant differences were observed in estrogen receptor expression, progesterone receptor expression, Ki-67 proliferation index, HER2 status, or tumor-infiltrating lymphocytes between baseline and post-stimulation biopsies.
The mean Ki-67 value was 41.3% at baseline and 40.2% after stimulation. This small difference was not statistically significant.
The investigators also assessed whether post-stimulation Ki-67 was linked to stimulation-related factors. No significant association was found with stimulation duration, total gonadotropin dose, or peak estradiol levels.
This finding is clinically important because Ki-67 is commonly used as a marker of tumor proliferation. The absence of a significant Ki-67 increase suggests that controlled ovarian stimulation with letrozole did not trigger measurable short-term proliferative activity in tumor tissue.
Estradiol Levels Did Not Translate Into Higher Proliferation
Although letrozole is used to reduce estrogen exposure during stimulation, some patients still reached higher estradiol levels.
In this study, three patients had peak estradiol levels above 1000 pg/mL on the day of ovulation trigger. Even in this subgroup, no significant increase in Ki-67 expression was observed after stimulation.
Across the full cohort, the mean estradiol level on the trigger day was 516.4 pg/mL. The authors noted that this was far lower than levels reported in prior studies of healthy oocyte donors undergoing ovarian stimulation without the same oncologic context.
The findings support the role of letrozole in limiting estrogen-related biological effects during fertility preservation protocols for patients with breast cancer.
Molecular Subtypes Remained Highly Stable
The PAM50 molecular subtype distribution also remained largely unchanged after stimulation.
Before and after controlled ovarian stimulation, the distribution was stable across luminal A, luminal B, HER2-enriched, basal-like, and normal-like subtypes. Only two borderline changes were observed: one tumor shifted from luminal A to luminal B, and another shifted from luminal B to luminal A.
Both changes occurred near classification thresholds and did not appear to reflect major biological reprogramming.
When PAM50 signatures and risk of recurrence scores were assessed as continuous variables, no statistically significant differences were found before and after ovarian stimulation.
Risk Scores Showed Only Minor Fluctuations
Five shifts in risk of recurrence category were observed. Four moved toward a lower-risk category, while one moved from intermediate to high risk.
The upward shift occurred in a HER2-enriched tumor and was described as minimal when evaluated on the continuous score. The authors emphasized that these changes were borderline and small in magnitude.
To better understand whether these variations might reflect stimulation-related effects or normal sampling variability, the researchers also evaluated paired diagnostic biopsy and surgical specimens from seven patients who did not receive neoadjuvant therapy.
In that exploratory analysis, some molecular variations were similar to or greater than those seen after ovarian stimulation. This suggests that the modest changes observed in the stimulation cohort may fall within the expected range of biological and technical variability between paired tumor samples.
Gene Expression Did Not Show Significant Stimulation-Driven Changes
The primary gene-expression analysis did not identify any significantly differentially expressed genes after ovarian stimulation when applying correction for multiple testing.
An exploratory analysis without correction showed downregulation of 11 of 72 genes in the overall cohort, including proliferation-associated genes such as CDC6, CENPF, EXO1, and RRM2. However, these changes did not meet the predefined threshold for statistical significance after false discovery rate correction.
In the hormone receptor-positive/HER2-negative subgroup, only limited gene-expression changes were observed.
Overall, the transcriptomic data did not indicate meaningful stimulation-induced activation of proliferation, estrogen signaling, or other tumor-associated molecular pathways.
Why These Findings Matter
Fertility preservation is often discussed under significant emotional and clinical pressure. Young patients must make decisions quickly, usually before starting chemotherapy or other systemic treatment.
For clinicians, one of the persistent concerns has been whether controlled ovarian stimulation could worsen tumor biology, especially in hormone receptor-positive breast cancer. While previous retrospective studies have not shown worse oncologic outcomes, direct biological evidence from tumor tissue has been limited.
This study adds an important layer of evidence because it examined the tumor itself before and after stimulation.
The findings suggest that controlled ovarian stimulation with letrozole does not produce measurable short-term changes in key pathological markers, proliferation, PAM50 subtype, or molecular recurrence risk.
A Reassuring Signal, With Limits
The study provides reassuring short-term data, but the authors also acknowledged several limitations.
The cohort was small, with 21 patients. It included different breast cancer subtypes, which may increase biological variability. Core biopsies also sample only part of a tumor, meaning some differences between paired samples may reflect intratumoral heterogeneity rather than treatment effect.
Another limitation is the short observation window. Post-stimulation biopsies were obtained within 24 to 48 hours after oocyte retrieval, so the study cannot fully assess delayed transcriptomic changes or long-term oncologic outcomes.
Longer follow-up and larger cohorts will be needed to determine whether these short-term biological findings translate into sustained clinical safety.
A Practical Message for Breast Cancer Fertility Preservation
For young women with breast cancer, fertility preservation can be a meaningful part of treatment planning. The ability to offer controlled ovarian stimulation without evidence of immediate tumor activation may help support timely, informed discussions between oncology and reproductive medicine teams.
In this prospective paired-biopsy study, controlled ovarian stimulation with concurrent letrozole was not associated with significant short-term histological or molecular changes in breast cancer tissue.
The data support the short-term biological safety of this approach and reinforce the importance of integrating fertility preservation counseling into breast cancer care for eligible young patients.
Written by Nare Hovhannisyan, MD
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