Christiani A . Amorim | UCLouvain (University of Louvain) (original) (raw)

Papers by Christiani A . Amorim

Journal of Assisted Reproduction and Genetics, Apr 20, 2023

Human Reproduction Update, Mar 14, 2022

Trabajo presentado en International Longevity and Cryopreservation Summit 2017, celebrado en Madr... more Trabajo presentado en International Longevity and Cryopreservation Summit 2017, celebrado en Madrid (Espana) del 25 al 27 de Mayo de 2017

Survival rates of many malignant diseases are steadily improving, but for patients of childbearin... more Survival rates of many malignant diseases are steadily improving, but for patients of childbearing age, fertility restoration often becomes a vital concern after disease remission. In women, treatments such as chemo/radiotherapy can be very harmful to the ovaries, causing loss of both endocrine and reproductive functions. When gonadotoxic treatment cannot be delayed, ovarian tissue cryobanking is the only way of preserving fertility. However, this technique is not advisable for patients with certain types of cancer, since there is a risk of reintroducing malignant cells present in the cryopreserved tissue. For these patients, a safer alternative could be transplantation of isolated preantral follicles back to their natural environment. To encapsulate and protect isolated follicles, a transplantable artificial ovary needs to be created. The main goal of the artificial ovary is to mimic the natural organ and for this, it should be composed of a matrix that encapsulates and protects not only the isolated follicles, but also autologous ovarian cells and bioactive factors, which are necessary for follicle survival and development. In this lecture, we will describe how to create an artificial ovary based on the natural ovarian follicle microenvironment. Additionally, we will discuss the indications, advantages and the different approaches to develop this new technology.

Because of the practical aspects of vitrification and its advantages for cryopreservation complex... more Because of the practical aspects of vitrification and its advantages for cryopreservation complex tissues, many authors have been studying it as an alternative way of cryopreserving ovarian tissue. In the last decade, many studies have been published on vitrification of ovarian tissue from both humans and animals. Different vitrification solutions and protocols, mostly adapted from embryo and oocyte vitrification, have been applied. The results have been discrepant from species to species and even within the same species, but lately they seem to indicate that vitrification can achieve similar or even superior results to conventional freezing. Despite the encouraging results obtained with vitrification of ovarian tissue from humans and different animal species, it is necessary to understand how vitrification solutions and protocols can affect ovarian tissue, notably preantral follicles. In addition, it is important to bear in mind that utilization of different approaches to assess tissue functionality and oocyte quality are essential in order to validate the promising results already obtained with vitrification procedures.

Objective: The aim of this study was to create an artificial ovary to provide an alternative way ... more Objective: The aim of this study was to create an artificial ovary to provide an alternative way of restoring fertility in patients who cannot undergo transplantation of cryopreserved ovarian tissue due to the threat of reintroducing malignant cells. Materials and methods: Ovaries from six-week-old female NMRI mice were removed for isolation of preantral follicles and ovarian cells (OCs). Thereafter, isolated follicles and OCs were encapsulated in two fibrin matrices containing low concentrations of fibrinogen (mg/ml) and thrombin (IU/mL) (F/T: F12.5/T1 and F25/T4) and autografted to mice. After one week, follicular density and development, OC survival and proliferation, inflammatory response and vascularization were evaluated. Results: After grafting, the follicle recovery rate ranged between 30.8% (F25/T4) and 31.8% (F12.5/T1). With both fibrin formulations, all follicles were found to be alive or minimally damaged, as demonstrated by TUNEL assay, and at the growth stage (primary, secondary and antral), confirmed by Ki67 immunostaining. Isolated OCs also survived and proliferated after grafting, as evidenced by less than 1% of apoptotic cells and a high proportion of Ki67-positive cells. Vessels were found in both fibrin formulations and the global vascular surface area varied from 1.35% (F25/T4) to 1.88% (F12.5/T1). Numerous CD45-positive cells were also observed in these two combinations. Conclusions: The present study is the first to show survival and growth of isolated murine ovarian follicles one week after autotransplantation of isolated OCs in a fibrin scaffold. The results indicate that fibrin is a promising candidate as a matrix for the construction of an artificial ovary. Xenotransplantation of isolated human follicles and OCs is the necessary next step to validate these findings.

In recent years, advanced chemo/radiotherapeutic treatments have led to high survival rates in ca... more In recent years, advanced chemo/radiotherapeutic treatments have led to high survival rates in cancer patients, giving rise to new issues for cancer survivors. Indeed, one major concern is future fertility in these women, since they may face premature ovarian failure. For this reason, different strategies have been proposed to preserve their fertility. When gonadotoxic treatment cannot be delayed, ovarian tissue cryobanking appears to be the most promising way of preserving a patient’s fertility. Moreover, this is the sole means of safeguarding fertility in prepubertal girls. Autotransplantation is the only option able to reestablish ovarian function from cryopreserved ovarian tissue in cancer survivors at present. So far, this technique has led to successful ovarian function restoration and up to 40 pregnancies in a number of centers around the world. However, there is a legitimate concern regarding the possible presence of malignant cells in frozen-thawed fragments, which could provoke a recurrence of the primary disease after reimplantation. Although many types of cancer never metastasise to the ovaries, leukaemia is systemic in nature and poses a greater threat to the patient, while breast cancer and some types of lymphoma are classed as moderate risk. For these patients, a safer alternative could be grafting of isolated preantral follicles, as these structures are enclosed in a basement membrane that prevents direct contact between follicular cells and capillaries, white blood cells, and nerve processes. Since ovarian cells (OCs) are essential for follicle development and neovascularization, autologous OCs should be grafted together with isolated follicles. To replace the original ovarian structure, a transplantable artificial ovary (TAO) should be created in order to encapsulate and protect the isolated follicles and OCs. As in case of a natural ovary, the main goal of the TAO is to offer an environment that allows follicle survival and development. Therefore, a TAO should maintain the original structure of follicles, ensure proper communication between follicles and OCs, preserve their interaction with the extracellular matrix and supply factors involved in follicular survival and development. In other words, the TAO should spatially and temporally mimic the ECM. In order to do so, it should include some design parameters, such as physical support of follicles, porosity, bioactivity, vascularization, interaction with cells, and biodegradability, which are all interconnected and influence each other.

Objective: The aim of this study was to evaluate the efficacy of a vitrification protocol develop... more Objective: The aim of this study was to evaluate the efficacy of a vitrification protocol developed by our group to cryopreserve ovarian tissue. For this, we used ovaries from non-human primates in order to have an animal model close to the clinical setting. Materials and methods: Ovarian biopsies from five adult baboons were vitrified, warmed and autografted. After five months, follicle survival, growth and function were assessed. The quality of stromal tissue and influence of the vitrification procedure on the cooling rate were also evaluated. Results: Our results showed that after vitrification, warming and grafting, follicles were able to grow and maintain their function, as illustrated by Ki67, anti-Mullerian hormone and growth differentiation factor-9 immunostainings. Corpora lutea were also observed, evidencing successful ovulation in all animals. Stromal tissue quality did not appear to be negatively affected by our cryopreservation procedure, as demonstrated by vascularization and proportions of fibrotic areas, which were similar to those found in fresh ungrafted ovarian tissue. Conclusion: Our results indicate that baboon ovarian tissue can be successfully cryopreserved using our vitrification protocol. However, before applying this technique in a clinical setting, we need to validate it by obtaining pregnancies.

Ovarian tissue cryopreservation and transplantation has been implemented worldwide as a routine p... more Ovarian tissue cryopreservation and transplantation has been implemented worldwide as a routine procedure to safeguard fertility in cancer patients. However, grafted ovarian fragments may have a very short lifespan due to the irregular distribution of primordial follicles and their significant loss after transplantation2. Furthermore, there are still concerns about its application in patients with certain types of disease because of the risk of cancer cell transmission3. An alternative would be grafting of isolated preantral follicles. This would allow the assessment of the follicular population before grafting, and the entire process of follicular development would occur in the graft recipient. Promising results obtained after isolation and xenografting of human preantral follicles embedded in plasma clot4,5,showed that this approach is feasible and encouraged us to pursue this path, focusing on the development of a scaffold that would mimic the ovary. Applying the principles of tissue engineering, we aim to design a scaffold to embed isolated follicles and act as a template to guide tissue growth up to its final form, allowing attachment, proliferation and migration of cells, angiogenesis and transport of molecules, oxygen and nutrients, and metabolic waste removal. In addition, it would have all the positive features of a plasma clot, such as fast degradation, biocompatibility and ability to maintain the original three-dimensional structure of follicles. Finally, as an off-the-shelf living tissue product, it would be easy to handle and graft. However, developing a scaffold that aims to recreate such a complex organ requires coordinated efforts of different professionals, including clinicians, engineers, biologists, chemists, surgeons and physicists, coupled with creative ideas on the relationship between follicular survival and development, material degradation and host response. Although this challenging and exciting approach is still in its infancy, we believe that, in the near future, it will become a new field in assisted reproductive technology.

A utilização e o desenvolvimento de biotécnicas da reprodução animal são condições indispensáveis... more A utilização e o desenvolvimento de biotécnicas da reprodução animal são condições indispensáveis para o aumento da eficiência produtiva dos rebanhos. Neste sentido, especialmente no tocante a ruminantes domésticos, biotécnicas como a inseminação artificial e a transferência de embriões vêm sendo utilizadas com sucesso. Outras biotécnicas terão sua aplicabilidade prática em larga escala no futuro. Nesse grupo, pode-se incluir a fecundação in vitro (FIV), clonagem e manipulação de oócitos inclusos em folículos ovarianos pré-antrais (MOIFOPA). No intuito de facilitar a compreensão da importância desta nova biotécnica, bem como, demonstrar como ela se inserirá no futuro, no contexto da reprodução animal, este capítulo será dividido em duas partes. A parte I será dedicada à uma breve revisão sobre os folículos ovarianos, enfatizando a sua formação, classificação e destino no interior dos ovários. Na parte II será abordada a importância do estudo da população de folículos ovarianos pré-antrais (FOPA) isolados in vitro, as principais técnicas de isolamento, conservação e cultivo de FOPA, o estado atual da MOIFOPA e, finalmente, serão discutidas as perspectivas de utilização da biotécnica de MOIFOPA na reprodução de animais domésticos, silvestres e em perigo de extinção

Human Reproduction, Apr 9, 2018

Do adipose tissue-derived stem cells (ASCs) enhance vascularization and follicle survival in xeno... more Do adipose tissue-derived stem cells (ASCs) enhance vascularization and follicle survival in xenografted ovarian tissue using a two-step transplantation approach? SUMMARY ANSWER: Higher rates of oxygenation and vascularization of ovarian tissue, as well as increased follicle survival rates, were detected in the early post-grafting period. WHAT IS KNOWN ALREADY: ASCs have multilineage differentiation potential, proangiogenic properties and enhance vascularization in a peritoneal grafting site. Some studies suggest that using ASCs may improve ovarian tissue quality by enhancing graft angiogenesis. STUDY DESIGN, SIZE, DURATION: A total of 15 severe combined immunodeficient (SCID) mice were intraperitoneally grafted with frozen-thawed human ovarian tissue (OT) from five different patients. A peritoneal transplantation site had been previously prepared in a first step using either empty fibrin (Fi+OT group [n = 5]) or ASC-loaded fibrin (Fi/ASCs+OT group [n = 5]) for 14 days prior to grafting. Five mice underwent the standard one-step transplantation procedure and served as controls (OT group). Lithium phthalocyanine (LiPc) crystals were inserted into all grafted human ovarian tissue before transplantation. Levels of partial pressure of oxygen (pO 2) in grafts were monitored in vivo by electron paramagnetic resonance (EPR) oximetry on Days 3 and 7. Samples for histology and immunohistochemistry (IHC) were collected after euthanizing the mice on Day 7 following EPR. One piece of ovarian tissue per patient was fixed for analysis to serve as non-grafted controls.

Obstetrical & Gynecological Survey, May 1, 2018

Ten percent to 20% of all ovarian epithelial tumors are borderline ovarian tumors (BOTs). These l... more Ten percent to 20% of all ovarian epithelial tumors are borderline ovarian tumors (BOTs). These low-grade malignancies rarely metastasize. Typically diagnosed at an early stage, BOTs usually have an excellent prognosis, but up to 4% of cases can evolve into more aggressive lesions. Approximately one third of patients with these tumors are under 40 years of age. The criterion standard treatment for BOTs is radical surgery with bilateral salpingo-oophorectomy. However, conservative surgery, either unilateral salpingo-oophorectomy or cystectomy, can be performed to preserve future fertility in women of reproductive age wishing to conceive. The risk of recurrence after radical surgery is low (0%-5%). Almost always relapses show BOT histology and can be safely managed with a second surgical intervention. A good option for patients who do not wish to conceive immediately is ovarian stimulation followed by oocyte or embryo cryopreservation. Ovarian tissue cryopreservation has proved to be a valuable strategy in young patients and those who cannot delay anticancer treatment. However, the risk of reintroducing malignant cells with ovarian tissue cryopreservation has been subject of debate for many years. Reimplanting ovarian tissue from acute leukemia patients has been deemed unsafe, whereas studies in other types of cancer (breast cancer and bone and soft tissue sarcoma) are reassuring. The aim of this prospective experimental study was to evaluate the safety of transplanting cryopreserved ovarian tissue from patients with BOTs. The study was conducted in an academic research unit. Ovarian cortical tissue samples from 11 patients undergoing cryopreservation for BOTs were evaluated. Samples of frozen-thawed ovarian tissue were analyzed using histology, immunohistochemistry (IHC) for mucin 1 (MUC1), and cytokeratin 7 (CK7) and molecular analysis by reverse transcription quantitative polymerase chain reaction (RT-qPCR) for CK7 and MUC1. Long-term (5 months) xenografting of ovarian tissue to 11 severe combined immunodeficient female mice was performed. Xenografts were analyzed by histology, IHC, and RT-qPCR. In addition, IHC for a highly specific marker of endometriosis, CD10, was performed on a selected sample. When analyzed by histology, IHC, and RT-qPCR, 10 of the 11 ovarian tissue samples were negative. Analysis of the 11 xenograft samples indicated that 9 were negative for malignant cells, but in 2 cases, glandular lesions were detected by histology. CK7 and MUC1 markers were demonstrated by IHC in these 2 xenografts as was CK7 expression by RT-qPCR. For the xenograft in which the original ovarian tissue was positive, there was confirmation of a BOT, whereas in the other case, expression of endometriosis marker CD10 was demonstrated by IHC. These data show that BOT cells are present in cryopreserved ovarian tissue from BOT patients. Therefore, preimplantation analysis of ovarian cortex is an absolute prerequisite. Because cryopreserved ovarian fragments cannot be tested before transplantation, there is no guarantee even with preimplantation analysis that all cryopreserved fragments will be free of BOT cells.

Journal of Assisted Reproduction and Genetics, Nov 15, 2019

Purpose Our aim was to elucidate the mechanisms involved in follicle activation of the ovarian re... more Purpose Our aim was to elucidate the mechanisms involved in follicle activation of the ovarian reserve after human ovarian tissue transplantation, with specific focus on the role of the effectors of the PI3K (mTOR and FOXO1) and Hippo (YAP) signaling pathways and whether they are somehow altered. Methods Frozen-thawed ovarian tissue was collected from six women (age 25-35 years) undergoing surgery for non-ovarian pathologies and divided into 4 fragments in each case: one for non-grafted controls and three for grafting to immunodeficient mice for 3, 7 and 21 days. The tissue was processed for hematoxylin and eosin staining, immunohistochemistry and immunofluorescence at different timepoints before and after grafting. Activation of the PI3K and Hippo signaling pathways was investigated by analysis of mTOR phosphorylation, FOXO1 cytoplasmic localization and YAP nuclear localization. Results No change in mTOR levels was observed in primordial follicles post-transplantation, but a significant upturn was recorded in growing follicles compared with primordial follicles, irrespective of grafting time. A higher percentage of primordial follicles was also found with FOXO1 in the cytoplasm after 3 days of transplantation than in non-grafted controls. Finally, a greater proportion of primordial follicles was detected with YAP in the nucleus at all timepoints after grafting. Conclusions This study supports the hypothesis that follicle activation may occur as an early event after transplantation, with follicle growth and death both contributing to the burnout phenomenon. This is the first time that the effectors of the PI3K and Hippo pathways have been investigated in grafted human ovarian tissue and their role in burnout documented.

Human Reproduction, Jul 1, 2021

Study question To investigate whether adipose tissue-derived stem cells (ASCs) modulate hypoxia a... more Study question To investigate whether adipose tissue-derived stem cells (ASCs) modulate hypoxia and oxidative stress in human ovarian tissue transplants to reduce early follicle loss. Summary answer ASCs protect the follicle pool by mitigating the hypoxia-related response through HIF1↑ signaling in human xenografts and enhancing revascularization by ensuring faster tissue reperfusion. What is known already ASCs are known for their angiogenic potential and capacity to boost angiogenesis by secreting growth factors and differentiating into vessels in numerous models of wound healing in regenerative medicine. In a 2-step ovarian tissue xenotransplantation involving grafting inside a fibrin scaffold two weeks prior to transplantation, ASCs reduced follicle loss after short- and long-term grafting, as well as abnormal follicle activation, by increasing reoxygenation and revascularization in human xenografts. Study design, size, duration Prospective experimental study. Cryopreserved ovarian cortex from five adult women was transplanted to 30 nude mice, with or without ASCs (ASC group; OT group). Ovarian grafts were retrieved on days 3 (n = 5), 10 (n = 5) and 21 (n = 5). One piece of ovarian tissue per patient was fixed for analysis after thawing to serve as non-grafted controls. Participants/materials, setting, methods The 10 animals grafted for 21 days underwent in vivo microdialysis evaluation to investigate direct reactive oxygen species (ROS) kinetics. Analyses of ovarian grafts at all time points and non-grafted controls included immunolabeling for double CD34 (revascularization by host and graft components), immunofluorescence for HIF1α (hypoxia-related response), Nrf2 (oxidative stress-related response) and 8OHdG (oxidative stress-related DNA damage), and gene expression (RT-qPCR) for VEGF-A (neoangiogenesis), SOD2 (antioxidant activity) and Nrf1 (mitochondrial biogenesis). Main results and the role of chance ROS peaked sooner in the ASC group (day 2, p &amp;lt; 0.0001) than the OT group (day 10, p = 0.01) after grafting, indicating earlier tissue reperfusion. Total vascularization was stable in the ASC group at all time points, but lower in the OT group 3 days after grafting (p = 0.01) due to a drop in both host and graft vascular components. HIF1α expression, detected mainly in follicles, was significantly lower in primordial follicles in the ASC group than the OT group on days 3 (p = 0.008) and 10 (p = 0.01). VEGF gene expression rose significantly (around 40x) in both groups on day 3 and persisted significantly longer in the ASC group (10 days) than the OT group (3 days) (p = 0.04), emphasizing the role of ASCs as enhancers of proangiogenic factors. There was no upturn in the oxidative stress-related response (Nrf2 pathway) nor DNA damage (8OHdG) to follicles in any of the grafted groups over time, while a modest increase in both markers was observed only in the stroma after 21 days. Neither was there any major increase in SOD2 and Nrf1 gene expression, suggesting no significant activation of the Nrf2 pathway for cytoprotection from oxidative stress. Limitations, reasons for caution Although Nrf2 signaling activation was detected in human granulosa cell cultures in increasing ROS concentrations, our findings did not confirm its role in tissue damage modulation after ovarian tissue transplantation. Further studies may evidence the involvement of other pathways that modulate oxidative stress after transplantation. Wider implications of the findings The role of ASCs in protecting the follicle pool appears to be related to a decrease in hypoxia and faster ovarian graft revascularization and reperfusion, sustained by an increase in VEGF for a longer period after grafting. There was no evidence of oxidative stress-related damage, irrespective of the transplantation strategy. Trial registration number

PubMed, May 22, 2020

To support survival and growth of follicles, the transplantable artificial ovary should mimic the... more To support survival and growth of follicles, the transplantable artificial ovary should mimic the original organ, offering a physical (3D matrix) and biological support (cells). In order to replicate the ovarian cell populations, the aim of this study is to assess the proportions of stromal and endothelial cells in the ovarian cortex. To this end, ovarian biopsies were obtained from six women (mean age: 49 years). The epithelial layer and medulla were carefully removed. The cortex was finely minced and enzymatically digested and the isolated cells were fixed. For cell characterization, immunostaining for CD31 (for endothelial cells) and inhibin-α (for granulosa cells) was performed. Positive cells in each staining were counted and the proportion of the different cell populations was estimated from the total number of isolated cells. Since there is no specific marker for ovarian stromal cells, we estimated the proportion of these cells by performing a vimentin immunostaining and subtracting the proportions of CD31- and inhibin-α-positive cells. Immunostaining showed that 84% of isolated cells were vimentin-positive. From this pool, 3% were endothelial cells and 1% granulosa cells. Consequently, the population of ovarian stromal cells was 80%. In conclusion, our findings show that stromal cells represent the larger population of cells in the human ovarian cortex. While this ensures follicle survival and development in a normal ovary, we believe that the low proportion of endothelial cells could have a negative impact on the angiogenesis in the artificial ovary after the first days of transplantation.

Journal of Assisted Reproduction and Genetics, Apr 20, 2023

Human Reproduction Update, Mar 14, 2022

Human Reproduction, Apr 9, 2018

Obstetrical & Gynecological Survey, May 1, 2018

Journal of Assisted Reproduction and Genetics, Nov 15, 2019

Human Reproduction, Jul 1, 2021

PubMed, May 22, 2020