Challenges in fertility preservation for patients with cervix cancer : clinical experiences and experimental developments

Cervical cancer is fairly prevalent among young women, with approximately one- third of patients being under 40 at the time of diagnosis. Despite most of cervical cancers in early stages can be resolved with conservative surgery including conization and trachelectomy, in more advanced stages, treatments including radiotherapy, chemotherapy and major surgery are required. These treatments will affect fertility chances and the opportunity to build a biologically related future family.

In Study I, the challenges that pose cervical cancer and its treatment to young women's fertility were explored in a prospective cohort of 91 patients diagnosed with stages I, II, III, or IV (67%, 25%, 7%, and 1%, respectively). Sixty-eight women underwent procedures for fertility preservation. During a follow-up of 8.3 years, 25 women (37%) returned to the center aiming at fertility treatments and five achieved conceptions either naturally or with assisted reproduction. This study highlights the significant gap in effective fertility preservation options for women diagnosed with cervical cancer.

In study II fertility-related distress was investigated in women treated for cervical cancer in the population-based Fex-Can prospective cohort. Women were classified according to maintenance or loss of uterine functionality due to the treatment received (PreservUt or LossUt, respectively) and questionnaires at study baseline approximately 1.5 years post diagnosis (T1) were collected, as well as during follow-up at 3- and 5-years post diagnosis (T2 and T3). After adjustment for sociodemographic covariates, women in the PreservUt group showed lower fertility-related distress in one of the six dimensions, partner disclosure, compared to LossUt group (adjusted OR 0.26, 95% CI 0.07-0.96) at T1. At T2, women in PreservUt group showed higher fertility related distress in becoming pregnant compared to LossUt group (adjusted OR, 11.6, 95% confidence interval CI 1.29-104.3). At T3, women in the PreservUt group were less likely (adjusted OR 0.10, 95% CI 0.17-0.57) as than those in the LossUt group to experience fertility related distress concerning partner disclosure. Conversely, women in PreservUt group were more likely (adjusted OR 5.00, 95% CI 1.02-24.7) than those in the LossUt group to report fertility-related distress concerning child's health at T3.

In study III and IV we explored experimental methods that could help developing novel treatments for patients losing endometrial and uterine functionality, a common consequence of the treatment of cervical cancer.

In Study III, the feasibility of endometrial tissue cryopreservation was evaluated using two methods: passive slow freezing (PSF) and vitrification (VT). Cells derived from both PSF and VT biopsies demonstrated sustained viability and retained their ability to generate functional epithelial organoids. However, post- thaw alterations were highlighted during histological assessments in epithelial cells in VT samples. Additionally, electron microscopy analysis revealed ultrastructural alterations in the mitochondria of both epithelial and stromal cells within the PSF samples. Overall, these findings suggest that despite minor ultrastructural changes, both PSF and VT techniques effectively preserved endometrial tissue morphology and maintained the ability to generate functional epithelial organoids.

In study IV, human endometrial organoids were generated to evaluate their hormonal responses along a 28 days cycle, modelling a menstrual cycle. Three hormone strategies were applied to stimulate human endometrial organoids: (1) estrogen (E) to replicate the proliferative phase, (2) Estrogen plus progesterone (EP) to induce the secretory phase (3) Estrogen, progesterone, and cAMP (EPC) to mimic the secretory phase in vivo. Gene and protein expression were thoroughly evaluated using RT-qPCR, IHC and ELISA to assess the organoids' hormonal responsiveness. The endometrial organoids exhibited appropriate hormonal responses, demonstrating increased expression of estrogen receptor (ESR1), progesterone receptor (PR), 17 ß -hydroxysteroid dehydrogenase (HSD17B1), Mucin 1 (MUC1), progesterone-associated endometrial protein (PAEP), and secreted phosphoprotein 1 (SPP1)-closely reflecting in vivo physiological processes. Notably, the most pronounced changes were detected in the EPC- treated hEOs at week 4, with significantly increased expression of glycodelin (PAEP) and osteopontin (SPP1), hallmark markers of the mid- to late-secretory phase. This organoid model effectively recapitulated the dynamic hormonal environment of the human menstrual cycle, providing a powerful platform for advancing research on endometrial disorders and supporting the development of personalized treatments in gynecology.

List of scientific papers

I. Marklund, A., Jiang, Y., Röjlar, H., Sergonioutis, F., Nilsson, H., Lundberg, F. E., & Rodriguez-Wallberg, K. A .. The complexity and challenges of fertility preservation in women with cervix cancer-A prospective cohort study reporting on reproductive outcome and overall survival. Acta Obstet Gynecol Scand. Nov 20 2024. https://doi.org/10.1111/aogs.15007

II. Jiang, Y., Rodriguez-Wallberg, K. A., Hellman K., Ullemar V., Wettergren L., Lampic C. Fertility-related distress following treatment for cervix cancer and the impact of maintaining or losing uterine functionality - a longitudinal population-based study. [Manuscript]

III. Saare, M .* , Wróbel, M .* , Jiang, Y.,* Rodriguez-Wallberg, K. A., Palomares, A. R., Kask, K., Kalinina, A., Apostolov, A., Minajeva, A., Kiisholts, K., Pathare, A. D. S., Laudański, P., Peters, M., & Salumets, A .. Biopsy vitrification: New tool for endometrial tissue cryopreservation for research applications. Cryobiology. Nov 14 2024;117:105161. *Equal contribution as first authors. https://doi.org/10.1016/j.cryobiol.2024.105161

IV. Jiang, Y., Palomares, A. R., Munoz, P., Nalvarte, I., Acharya, G., Inzunza, J., Varshney, M., & Rodriguez-Wallberg, K. A .. Proof-of-Concept for Long-Term Human Endometrial Epithelial Organoids in Modeling Menstrual Cycle Responses. Cells. Nov 2 2024;13(21). https://doi.org/10.3390/cells13211811