Maria Macciocca

Cryopreservation of Ovarian Tissue in Pediatric Patients

Cancer treatments improve the survival rate of children and adolescents; however chemo- and radiotherapy result in gonadal damage leading to acute ovarian failure and sterility. Ovarian tissue cryopreservation allows long-term storage of primordial follicles and represents the only possibility of preserving the potential fertility in prepubertal girls. The aim of the present study is to describe our experience in ovarian tissue cryopreservation in 45 pediatric patients. The number of follicles per square millimeter of the overall section area and follicle quality were evaluated histologically. A strong negative correlation was found between age and follicular density in patients both prior to and after chemotherapy (P < 0.0001). Damage in follicular quality, that is, increased oocyte vacuolization and detachment of the oocyte from granulosa cells, was found after chemotherapy. Ovarian tissue cryopreservation, preferably performed before initiation of chemotherapy, should be offered to pediatric patients, including prepubertal girls, at risk of sterility.

Good Preservation of Stromal Cells and No Apoptosis in Human Ovarian Tissue after Vitrification

The aim of this study was to develop a vitrification procedure for human ovarian tissue cryopreservation in order to better preserve the ovarian tissue. Large size samples of ovarian tissue retrieved from 15 female-to-male transgender subjects (18–38 years) were vitrified using two solutions (containing propylene glycol, ethylene glycol, and sucrose at different concentrations) in an open system. Light microscopy, transmission electron microscopy, and TUNEL assay were applied to evaluate the efficiency of the vitrification protocol. After vitrification/warming, light microscopy showed oocyte nucleus with slightly thickened chromatin and irregular shape, while granulosa and stromal cells appeared well preserved. Transmission electron microscopy showed oocytes with slightly irregular nuclear shape and finely dispersed chromatin. Clear vacuoles and alterations in cellular organelles were seen in the oocyte cytoplasm. Stromal cells had a moderately dispersed chromatin and homogeneous cytoplasm with slight vacuolization. TUNEL assay revealed the lack of apoptosis induction by vitrification in all ovarian cell types. In conclusion after vitrification/warming the stromal compartment maintained morphological and ultrastructural features similar to fresh tissue, while the oocyte cytoplasm was slightly damaged. Although these data are encouraging, further studies are necessary and essential to optimize vitrification procedure.

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.

Cryopreservation of ovarian tissue in breast cancer patients: 10 years of experience

AIM To present a decade of experience with ovarian tissue cryopreservation in breast cancer patients. MATERIALS & METHODS The safety of the procedure was histologically evaluated before and after freezing in 94 patients. Out of 94 patients, 48 prechemotherapy patients were randomly selected to determine stroma and follicle preservation and follicular density. RESULTS The ovarian tissue from 94 patients did not identify any micrometastases. After cryopreservation, morphology of the ovarian tissue and density of healthy follicles were similar in fresh and frozen tissue. Follicular density decreased with the increasing age of patients in both fresh and frozen tissue (p < 0.0001). A variation in follicular density was observed between fresh and frozen tissue (p < 0.05). CONCLUSION These results suggest that ovarian tissue cryopreservation is highly feasible for preserving the fertility of young breast cancer patients.

Collagen fibre arrangement and functional crimping pattern of the medial collateral ligament in the rat knee

Ligaments have been described as multifascicular structures with collagen fibres cross-connecting to each other or running straight and parallel also showing a waviness or crimping pattern playing as a shock absorber/recoiling system during joint motions. A particular collagen array and crimping pattern in different ligaments may reflect different biomechanical roles and properties. The aim of the study was to relate the 3D collagen arrangement in the crimping pattern of the medial collateral ligament (MCL) to its functional role. The MCL is one of the most injured ligaments during sports activities and an experimental model to understand the rate, quality and composition of ligaments healing. A deep knowledge of structure–function relationship of collagen fibres array will improve the development of rehabilitation protocols and more appropriate exercises for recovery of functional activity. The rat MCL was analysed by polarized light microscopy, confocal laser microscopy and scanning electron microscopy (SEM). Histomorphometric analysis demonstrated that MCL crimps have a smaller base length versus other tendons. SEM observations demonstrated that collagen fibres showing few crimps were composed of fibrils intertwining and crossing one another in the outer region. Confocal laser analyses excluded a helical array of collagen fibres. By contrast, in the core portion, densely packed straight collagen fibres ran parallel to the main axis of the ligament being interrupted both by planar crimps, similar to tendon crimps, and by newly described right-handed twisted crimps. It is concluded that planar crimps could oppose or respond exclusively to tensional forces parallel to the main ligament axis, whereas the right-handed twisted crimps could better resist/respond to a complex of tensional/rotational forces within the ligament thus opposing to an external rotation of tibia.

Contribution of glycosaminoglycans to the microstructural integrity of fibrillar and fiber crimps in tendons and ligaments.

The biomechanical roles of both tendons and ligaments are fulfilled by the extracellular matrix of these tissues. In particular, tension is mainly transmitted and resisted by protein (collagen, elastin) fibers, whereas compression is opposed by water-soluble glycosaminoglycans (GAGs). GAGs spanning the interfibrillar spaces and interacting with fibrils through the interfibrillar proteoglycans also seem to play a part in transmitting and resisting tensile stresses. Both tendons and ligaments showing similar composition, but different functional roles and collagen array, exhibit periodic undulations of collagen fibers or crimps. Each crimp is composed of many knots of each single fibril or fibrillar crimps. Fibrillar and fiber crimps play a mechanical role in absorbing the initial loading during elongation of both tendons and ligaments, and in recoiling fibrils and fibers when tissues have to return to their original length. This study investigated whether GAGs covalently attached to proteoglycan core proteins directly affect the 3D microstructural integrity of fibrillar crimp regions and fiber crimps in both tendons and ligaments. Achilles tendons and medial collateral ligaments of the knee from eight female Sprague-Dawley rats (90 days old) incubated in a chondroitinase ABC solution to remove GAGs were observed under a scanning electron microscope (SEM). In addition, isolated fibrils of these tissues obtained by mechanical disruption were analyzed by a transmission electron microscope (TEM). Both Achilles tendons and medial collateral ligaments of the rats after chemical or mechanical removal of GAGs still showed crimps and fibrillar crimps comparable to tissues with a normal GAG content. All fibrils in the fibrillar crimp region always twisted leftwards, thus changing their running plane, and then sharply bent, changing their course on a new plane. These data suggest that GAGs do not affect structural integrity or fibrillar crimp functions that seem mainly related to the local fibril leftward twisting and the alternating handedness of collagen from a molecular to a supramolecular level.

Confocal laser scanning microscopy analysis of bioenergetic potential and oxidative stress in fresh and frozen-thawed human ovarian tissue from oncologic patients

OBJECTIVE To evaluate the effectiveness of a bioenergy/oxidative stress assessment based on confocal laser scanning microscopy (CLSM) in association with morphology and ultrastructure analyses based on light microscopy (LM) and transmission electron microscopy (TEM), to monitor the preservation status of cryopreserved human ovarian tissue from cancer patients. DESIGN Prospective study. SETTING University hospital. PATIENT(S) Fourteen young cancer patients. INTERVENTION(S) Human ovarian tissue biopsy, slow freezing/rapid thawing, LM, TEM, CLSM assessment of mitochondrial distribution and activity, and intracellular reactive oxygen species (ROS) localization and levels. MAIN OUTCOME MEASURE(S) In tissue examined before and after slow freezing/rapid thawing, follicular and stromal LM-based score of morphologic damage, ultrastructure, mitochondrial distribution pattern, reactive oxygen species (ROS) localization; mean ± standard deviation of stromal mitochondrial activity and ROS levels. RESULT(S) Severe (n = 6 patients), slight (n = 6 patients), or no (n = 2 patients) LM/TEM-based damage was found in fresh tissue. After freezing/thawing, no further morphologic/ultrastructural alterations were found; however, statistically significant reductions, increases, or no changes in mitochondrial activity and ROS levels were found in severely, slightly, and undamaged tissue, respectively. CONCLUSION(S) Bioenergy/oxidative functional damage was found in tissue with severe LM/TEM-assessed damage. In tissue with slight LM/TEM-assessed damage, the CLSM-based bioenergy/oxidative stress assessment was the only test that allowed discrimination between tissue that had been better (low/no difference) or worse preserved (significant differences).

Effects of N-acetylcysteine on human ovarian tissue preservation undergoing cryopreservation procedure.

The aim of the study was to evaluate the effects of the antioxidant N-acetylcysteine (NAC), added in freezing/thawing solutions, on reactive oxygen species (RRS) levels and on ovarian tissue preservation after cryopreservation. Ovarian samples from 10 subjects suffering from cancer diseases were cryopreserved using the slow freezing/rapid thawing standard protocol without or with NAC supplementation. RRS levels produced during cryopreservation were monitored by electron paramagnetic resonance (EPR) spectroscopy. The preservation of fresh ovarian tissue (t0), thawed tissue (t1 and t1 NAC) and thawed tissue maintained at 4°C for 2 hrs (t2 and t2 NAC) was analysed by light microscopy, transmission electron microscopy, Ki67 immunohistochemical and TUNEL analysis. It was possible to design a maximum peak for RRS production at t1, which slightly decreased at t2. NAC reduced the extent of RRS levels in cryopreserved ovarian tissues if compared with non-supplemented ones, although not restoring RRS production to baseline values. Comparative analysis between the two cryopreservation protocols showed that a better preservation of morphological characteristics, proliferation index and DNA integrity of ovarian tissue was obtained using NAC and no differences between t1NAC and t2NAC were observed. The employment of NAC during cryopreservation procedure could be an useful strategy for preserving the function of endogenous cellular systems. Nevertheless, further studies on the viability of thawed ovarian tissue are needed to support the feasibility of this approach in clinical settings.

Effects of cyclic increase in gonadotropins on the in vitro development of primordial follicles to antral stage.

The aim of this study was to evaluate the effects of FSH and LH on follicle development during a long-term culture of cryopreserved human ovarian tissue, using morphological and ultrastructural examinations. Thawed ovarian tissue slices from a 4-year-old child with Wilms tumor were cultured for 32 weeks in two different culture conditions, without (medium A) and with (medium B) a monthly peaked increase in FSH and LH. At week 32, in the medium B cultured tissue, a cluster of preantral follicles associated with two oocytes prematurely ovulated was observed, suggesting that the cyclic increase of gonadotropins promoted thawed follicles to grow up to the antral stage. However, the integrity and coordinated follicle development were not maintained. Indeed, ultrastructural analysis showed a well-preserved “naked” oocyte with concomitant features of immaturity and maturity, as if this culture condition had led to an asynchronous maturation of oocyte cytoplasmic components.

High cytokine expression and reduced ovarian reserve in patients with Hodgkin lymphoma or non-Hodgkin lymphoma

OBJECTIVE To investigate the ovarian reserve in female lymphoma patients and the potential relationships with the cytokine network. DESIGN Age-matched control study. SETTING Womens university hospital. PATIENT(S) Seventy-three lymphoma patients (57 with classic Hodgkin lymphoma [HL] and 16 with non-Hodgkin lymphoma [NHL]), approaching our center for ovarian tissue cryopreservation (study group) were compared with 25 age-matched healthy volunteers (control group). INTERVENTION(S) Measurements of antimüllerian hormone (AMH), soluble interleukin-2 receptor (SIL-2R), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor α (TNF-α) levels. MAIN OUTCOME MEASURE(S) The AMH and cytokine levels of the lymphoma patients and the healthy volunteers were compared. Correlations between AMH with SIL-2R, IL-6, and IL-8 levels were performed. RESULT(S) The AMH showed significant lower concentrations in lymphoma patients than in the control group. Higher significant concentrations in lymphoma patients than in control group were found for SIL-2R and IL-6. No differences were observed comparing HL and NHL groups and within the stages of HL group for AMH and all the cytokines analyzed. Finally, significant inverse correlations were observed in lymphoma patients between AMH and SIL-2R, IL-6, and IL-8 levels, but not with TNF-α levels. Positive correlations between SIL-2R with IL-6, and IL-6 with IL-8 were also shown. CONCLUSION(S) In patients with HL or NHL at baseline the cytokine network is particularly active and the ovarian reserve is reduced. A strong negative correlation between AMH and SIL-2R, IL-6, and IL-8 has been also evidenced.