Effect of chemotherapy and antioxidants on the preservation of ovarian tissue of cancer patients undergoing cryopreservation

Introduzione La crioconservazione di tessuto ovarico rappresenta una valida strategia per preservare la funzione ovarica delle pazienti oncologiche a rischio di fallimento ovarico a causa dei trattamenti chemioterapici. Obiettivi 1. Valutare l’effetto dell’antiossidante N-acetilcisteina (NAC) sulle caratteristiche morfo-funzionali del tessuto ovarico sottoposto a crioconservazione. 2. Valutare in-vitro l’effetto protettivo di NAC ed ormone luteinizzante (LH) sul tessuto ovarico trattato con doxorubicina e cisplatino. Materiali e metodi 1. Il tessuto ovarico di 10 pazienti è stato crioconservato in presenza/assenza di NAC per valutare: a)i livelli di specie radicaliche reattive (SRR) prodotte durante la procedura di crioconservazione; b)la preservazione del tessuto ovarico crioconservato. 2. Cellule stromali, isolate da tessuto ovarico crioconservato di 5 pazienti, sono state trattate con doxorubicina e cisplatino per valutare: a)vitalità cellulare; b)attivazione dei processi apoptotici; c)inibizione di proliferazione e differenziamento cellulare. Successivamente le cellule stromali sono state trattate con doxorubicina e cisplatino in combinazione con NAC per valutare l’integrità cellulare e l’espressione di markers infiammatori, o in combinazione con LH per valutare la vitalità cellulare. Risultati e Conclusioni 1. La NAC determina una buona preservazione del tessuto ovarico sottoposto a crioconservazione e una riduzione dello stress ossidativo, sebbene non a livelli basali. Ulteriori studi sono necessari per testare concentrazioni di NAC più efficaci oppure per individuare altri agenti antiossidanti per ridurre i livelli di SRR. 2. Doxorubicina e cisplatino riducono la crescita cellulare, attivano l’apoptosi ed inibiscono la proliferazione e il differenziamento cellulare. Il cotrattamento delle cellule stromali con chemioterapici e NAC o LH riduce l’effetto citotossico dei farmaci migliorando la preservazione cellulare. Ulteriori studi sono necessari per poter collocare queste sostanze nella categoria di “fertisave agents” e poterle prescrivere alle pazienti in combinazione con il trattamento chemioterapico. La ricerca in questo campo dovrebbe continuare al fine di individuare altre sostanze efficaci nel proteggere le ovaia.Introduction The cryopreservation of ovarian tissue is a viable strategy for preserving ovarian function of cancer patients at risk of ovarian failure due to chemotherapy treatments. Aims 1.To evaluate the effect of antioxidant N-acetylcysteine (NAC) on the morpho-functional characteristics of the ovarian tissue undergoing cryopreservation. 2.To evaluate the protective in-vitro effect of NAC and luteinizing hormone (LH) on the ovarian tissue treated with doxorubicin and cisplatin. Materials and methods 1.The ovarian tissue of 10 patients was cryopreserved in presence/absence of NAC to assess: a)the levels of reactive radical species (SRR) produced during the cryopreservation procedure; b)the morphological preservation of cryopreserved ovarian tissue. 2.The stromal cells, isolated from cryopreserved ovarian tissue of 5 patients, were treated with doxorubicin and cisplatin to assess: a)cell viability; b)activation of the apoptotic processes; c)inhibition of cell proliferation and differentiation. Subsequently, the stromal cells were treated with doxorubicin and cisplatin in combination with NAC to evaluate the cellular integrity and the expression of inflammatory markers, or in combination with LH to assess cell viability. Results and Conclusions 1.The NAC determines a good preservation of cryopreserved ovarian tissue and a reduction of oxidative stress, although not at baseline levels. Further studies are needed to test the most effective concentrations of NAC or to identify other antioxidants to reduce the levels of SRR. 2.Doxorubicin and cisplatin reduce cell growth, activate apoptosis and inhibit cell proliferation and differentiation. The cotreatment of stromal cells with chemotherapeutic agents and NAC or LH reduces the cytotoxic effect of drugs by improving the cellular preservation. Further studies are needed to place these substances in the category of "fertisave agents" and to be able to prescribe them to patients in combination with chemotherapy. Research in this field should continue in order to identify other substances which are effective in the ovarian protection

Autotransplantation of cryopreserved ovarian tissue in oncological patients: recovery of ovarian function

AIM: To present preliminary results of autotransplantation of cryopreserved ovarian tissue performed at Sant'Orsola-Malpighi Hospital, Bologna, Italy. MATERIALS & METHODS: Orthotopic transplantation was performed in two women with colorectal and breast cancer, and heterotopic transplantation was performed in one Hodgkin's lymphoma woman. The presence of micrometastasis in the ovarian tissue was checked, and morphological features of ovarian tissue were evaluated before transplantation. Ovarian function was monitored by hormonal and ultrasound-color Doppler examination after transplantation. RESULTS: In all three women, no micrometastasis was found; light and transmission electron microscopy showed well-preserved thawed ovarian tissue. Ovarian function recovery was observed 2-4 months after transplantation. Spontaneous menstrual cycles occurred in two women with normal follicular densities. No periods occurred in the woman with low follicular density at the time of tissue collection. CONCLUSION: Ovarian tissue cryopreservation and transplantation is a promising approach for preserving ovarian function in women with cancer

A Wide Range of 3243A>G/tRNALeu(UUR) (MELAS) Mutation Loads May Segregate in Offspring through the Female Germline Bottleneck

Segregation of mutant mtDNA in human tissues and through the germline is debated, with no consensus about the nature and size of the bottleneck hypothesized to explain rapid generational shifts in mutant loads. We investigated two maternal lineages with an apparently different inheritance pattern of the same pathogenic mtDNA 3243A>G/tRNALeu(UUR) (MELAS) mutation. We collected blood cells, muscle biopsies, urinary epithelium and hair follicles from 20 individuals, as well as oocytes and an ovarian biopsy from one female mutation carrier, all belonging to the two maternal lineages to assess mutant mtDNA load, and calculated the theoretical germline bottleneck size (number of segregating units). We also evaluated “mother-to-offspring” segregations from the literature, for which heteroplasmy assessment was available in at least three siblings besides the proband. Our results showed that mutation load was prevalent in skeletal muscle and urinary epithelium, whereas in blood cells there was an inverse correlation with age, as previously reported. The histoenzymatic staining of the ovarian biopsy failed to show any cytochrome-c-oxidase defective oocyte. Analysis of four oocytes and one offspring from the same unaffected mother of the first family showed intermediate heteroplasmic mutant loads (10% to 75%), whereas very skewed loads of mutant mtDNA (0% or 81%) were detected in five offspring of another unaffected mother from the second family. Bottleneck size was 89 segregating units for the first mother and 84 for the second. This was remarkably close to 88, the number of “segregating units” in the “mother-to-offspring” segregations retrieved from literature. In conclusion, a wide range of mutant loads may be found in offspring tissues and oocytes, resulting from a similar theoretical bottleneck size

Cost-effectiveness analysis of ovarian tissue cryopreservation and transplantation for preservation of fertility in post-pubertal oncological women submitted to high-risk gonadotoxic chemotherapy

Objective: To study the economic impact of ovarian tissue cryopreservation and transplantation (OTC) in post-pubertal patients who underwent high-risk gonadotoxic chemotherapy. Methods: A decision tree model was used to determine the live birth rate and cost-effectiveness of OTC versus non-OTC. The incremental cost-effectiveness ratio (ICER) was calculated. A sensitivity analysis was performed under the assumption that the costs of ovarian cortex retrieval, cryopreservation, and storage for patients with cancer might be covered by the national health system or health insurance. Results: Patients had the greatest probability of achieving live birth after high-risk chemotherapy when they underwent OTC versus non-OTC. Although cryopreservation of ovarian tissue results in higher live birth rates, it is always more expensive. Cost-effectiveness increases when the majority of patients completes the path of tissue cryopreservation plus transplantation after 5 years. Conclusion: Although OCT has been demonstrated as a procedure for effective fertility preservation in fertility-age women with cancer, no cost-effectiveness analysis has been performed until now. This model could help healthcare systems to allocate coverage for OCT

Ovarian tissue cryopreservation and transplantation: 20 years experience in Bologna University

Objective: To report the 20-year experience in ovarian tissue cryopreservation (OTC) and ovarian tissue transplantation (OTT) of the Bologna clinical center (Bologna, Italy). Design: Retrospective cohort study. Patients: 1026 pediatrics and women aged between 2 and 38 years who underwent OTC and OTT between January 2002 to January 2022. Results: Of the 1026 patients, 238 (22.8%) were pediatrics (≤ 17 years, Group 1) and 788 (77.2%) were adult women (range 18-38 years, Group 2). In Group 1, 184 (77.3%) patients had malignant diseases and 54 (22.7%) had non-malignant diseases. In Group 2, 746 (94.7%) patients had malignant diseases and 42 (5.3%) had non-malignant diseases. No real complications were observed during surgery. In all the samples analyzed most of the follicles were in the resting stage, while only a few follicles were growing. In both fresh and thawed samples, follicular density was higher in Group 1 than in Group 2 (p < 0.01). Regardless of age, good preservation of follicles and stroma was observed in fresh and thawed ovarian tissue by histological and immunohistochemical analyses (estrogen and progesterone receptors; Ki67 and Bcl2 markers; TUNEL). To date, out of 1026 total women, 812 (79.1%) had their tissue stored. Sixty-eight (6.6%) patients died from their primary disease. Twentyfour (2.3%) women performed 33 OTTs between December 2011 and January 2022. Restoration of menstruation was observed in 15 out of 17 menopausal women. Six pregnancies were achieved, two hesitated in abortion and four in the birth of healthy babies. Conclusion: OTC is the only fertility preservation technique applicable in prepubertal/ pediatrics and in adult patients when stimulation for oocytes/embryos cryopreservation is not possible. The reported data can help future patients and physicians in their discussions and decisions about the need and possibilities of preserving ovarian function

Crioconservazione degli ovociti

In seguito all’entrata in vigore in Italia della legge 40/2004 che vieta il congelamento degli embrioni sviluppati in programmi di fertilizzazione in vitro e embrio-transfer (FIV-ET), la crioconservazione degli ovociti rappresenta l’unica alternativa per utilizzare al massimo una stimolazione ovarica: gli ovociti sopranumerari possono essere, infatti, congelati per evitare alla paziente di sottoporsi a cicli successivi di induzione dell’ovulazione multipla. La crioconservazione degli ovociti è inoltre particolarmente utile per le pazienti a rischio di iperstimolazione ovarica in cui è sconsigliabile il trasferimento embrionale su ciclo “fresco”: gli ovociti possono essere congelati ed utilizzati in un successivo ciclo differito. La crioconservazione degli ovociti permette infine di preservare la fertilità di donne a rischio di fallimento ovarico precoce per cause genetiche, patologie pelviche, come cisti ovariche, endometriosi, infezioni ricorrenti, o in seguito a trattamenti chemio- e radioterapici che determinano la distruzione del patrimonio germinale. Inizialmente, la crioconservazione dei gameti femminili ha incontrato notevoli difficoltà tecniche a causa delle caratteristiche peculiari e dell’alto contenuto di acqua dell’ovocita. Al fine di ottimizzare i risultati clinici, nel corso degli anni, sono stati messi a punto diversi protocolli e metodiche di crioconservazione che hanno portato nell’ultimo decennio ad un notevole incremento delle nascite. La crioconservazione degli ovociti rappresenta ad oggi una procedura fondamentale nelle tecniche di riproduzione assistita (ART) e non dovrebbe essere più considerata una tecnica sperimentale. In questo capitolo è riportata una revisione sistematica dei più importanti studi presenti in letteratura sulla crioconservazione degli ovociti evidenziando i progressi dei risultati clinici e le caratteristiche tecniche delle due procedure attualmente disponibili: il congelamento lento/scongelamento rapido (slow freezing/rapid thawing, SF/RT) e la vitrificazione/riscaldamento (vitrification/warming, V/W)

Transplantation of cryopreserved ovarian tissue in a patient affected by metastatic struma ovarii and endometriosis

In this case report, the outcomes of cryopreserved ovarian tissue transplantation performed in a patient affected by struma-ovarii associated with mature cystic teratoma, recurrent endometriotic cysts and diffuse peritoneal malignant struma-ovarii implants were described. Before cryopreservation, the patient underwent two left ovarian surgeries for enucleation cysts 8â\u80\u89years after righ salpingo-oophorectomy for struma-ovarii. Ovarian biopsy was collected in another hospital and transported to our laboratory for cryopreservation. The patient was submitted to radioiodine-therapy for metastases from malignant struma-ovarii. After treatment she experienced premature ovarian failure. Ten years after cryopreservation, a first orthotopic transplantation was performed in the left ovary and in a peritoneal pocket. Before transplantation, ovarian samples were analyzed to assess neoplastic contamination and tissue quality. Three years later, a second transplantation was heterotopically performed in abdominal subcutaneous sites. The analysis on thawed ovarian tissue did not reveal micrometastasis and they showed follicle and stroma damages. After transplantation few small follicles were observed at ultrasound examination and hormonal levels remained at menopausal values. To date no ovarian function recovery has been observed. The report highlights that ovarian tissue cryopreservation after multiple ovarian surgery may have some limitations. An accurate counseling should be offered to patients who wish to preserve fertility