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Purpose Exogenous gonadotrophins administration during in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles could significantly alter the endogenous follicular regulation system and could influence oocyte quality. The analysis of the follicular fluid (FF) cytokine and hormone profiles in physiological natural cycles is crucial to appreciate the role of FF milieu on follicle development. So far, the FF cytokine profile has been analyzed only in controlled ovarian stimulation cycles and in modified natural cycles. Our study defines, in physiological natural cycles, the cytokine and hormone profiles of individual FF aspirated from antral follicles. Methods A total of 203 FFs obtained from 83 women with regular menstrual cycles undergoing ovarian tissue cryopreservation were analyzed: 115 FFs from Group 1 (10 to 29 years of age) and 88 FFs from Group 2 (30 to 40 years of age). In individual FF, 27 cytokines were measured with xMAP technology, and progesterone, estrone, estradiol, testosterone, androstenedione concentrations were determined by liquid chromatography–tandem mass spectrometry. Results FF hormone profiles were not different in follicular and luteal phase, suggesting that FF hormones are regulated independently of the endogenous gonadotrophins—possibly because 74% of the punctured follicles, which were ≤6 mm, did not require cyclic pituitary function. The follicle size was influenced not only by the FF cytokine profile but also by the FF hormone profile, both of which are dependent on age. Main Conclusions In physiological natural cycles, FF hormones seems to be regulated independently of the endogenous gonadotropins. Age influences FF hormone and cytokine profiles and the compelling relationship between FF hormones and FF cytokines could influence the follicle development.

Diagnostic imaging has significantly grown over the last thirty years as indispensable support for diagnostic, prognostic, therapeutic and monitoring procedures of human diseases. This study explored the effects of low-dose X-ray medical diagnostics exposure on female fertility. To aim this, cumulus-oocyte complexes (COCs) recovered from the ovaries of juvenile sheep and human ovaries were used as complementary models for in vitro studies. In the sheep model, the effects of low-dose X-rays on oocyte viability and developmental competence were evaluated. In human ovaries originated from two age group (21–25 and 33–36 years old) subjects with gender dysphoria, X-rays effects on tissue morphology, follicular density and expression of apoptosis-related (NOXA, PUMA, Bcl2, Bak, γH2AX) and cell cycle-related genes (p21 and ki67) were investigated. It was noted that in sheep, the minimum dose of 10 mGy did not influence most of examined parameters at oocyte and embryo levels, whereas 50 and 100 mGy X-ray exposure reduced oocyte bioenergetic/oxidative activity but without any visible effects on oocyte and embryo development. In addition, blastocyst bioenergetic/oxidative status was reduced with all used doses. Overall data on human ovaries showed that low-dose X-rays, similarly as in sheep, did not alter any of examined parameters. However, in women belonging to the 33–36 year group, significantly reduced follicular density was observed after exposure to 50 and 100 mGy, and increased NOXA and Bax expression after exposure at 50 mGy. In conclusion, used low-doses of X-ray exposure, which resemble doses used in medical diagnostics, produce weak damaging effects on female fertility with increased susceptibility in advanced age.

In this case report, we describe the outcomes of two heterotopic transplantations of cryopreserved ovarian tissue performed in a patient with HL, after 11 and 15 years of storage. At the age of 30, the patient underwent laparoscopy to collect ovarian tissue for cryopreservation before chemotherapy and radiotherapy. Eleven years later she experienced premature ovarian failure (POF). As the patient was only interested in endocrine function recovery, two heterotopic ovarian tissue transplantations were performed in the abdominal wall above the rectus muscle respectively 11 and 15 years after cryopreservation. Before transplantation, ovarian samples were analyzed to assess neoplastic contamination and tissue quality. The analysis on thawed ovarian tissue did not reveal micrometastasis and it showed well-preserved follicles and stroma. After both ovarian tissue grafting, menopausal symptoms ceased. The patient had periods approximately every 30-days and hormonal levels were within the premenopausal range. The endocrine function lasted 3-years after the first heterotopic transplantation and is still ongoing after second transplantation. Cryopreservation of ovarian tissue should be proposed to HL patients, as the incidence of POF as a long-term complication is not negligible. In these patients heterotopic transplantation is a useful tool to eliminate menopausal symptoms, preventing osteoporosis and reducing cardiovascular risks.

This case report describes the first Italian live birth obtained by cryopreserved ovarian tissue transplantation in a woman affected by non-Hodgkin's lymphoma. Before anticancer treatments, several fertility preservation options were proposed. At 29 years the patient underwent laparoscopy for ovarian tissue cryopreservation. After treatments she experienced premature ovarian failure (POF) and asked for cryopreserved ovarian tissue transplantation. Before transplantation, ovarian samples were analyzed to assess neoplastic contamination and tissue quality. Two subsequent ovarian tissue transplantations were performed 4 and 7 years after cryopreservation. The follicle-stimulating hormone and luteinizing hormone reduction, estradiol increase and first menstrual cycle appeared 2 months after the second transplantation. The woman conceived spontaneously 5 months after the second transplantation. After 39 weeks of uneventful gestation, a healthy male baby was born. Ovarian tissue cryopreservation, thawing and transplantation successfully restored ovarian function and fertility after tissue storage.

INTRODUCTION: Ovarian tissue is increasingly being collected from cancer patients and cryopreserved for fertility preservation. Alternately to the autologous transplantation, the development of culture systems that support oocyte development from the primordial follicle stage represent a valid strategy to restore fertility. The aim of this study is to review the most recent data regarding oogenesis in vitro and to provide an up-to-date on the contemporary knowledge of follicle growth and development in vitro. EVIDENCE ACQUISITION: A comprehensive systematic MEDLINE search was performed since February 2018 for English-language reports by using the following terms: "ovary," "animal and human follicle," "in vitro growth and development," "ovarian tissue culture," "fertility preservation," "IVM," "oocyte." Previous published reviews and recent published original articles were preferred in order to meet our study scope. EVIDENCE SYNTHESIS: Over time, many studies have been conducted with the aim to optimize the characteristics of ovarian tissue culture systems and to better support the three main phases: 1) activation of primordial follicles; 2) isolation and culture of growing preantral follicles; 3) removal from the follicle environment and maturation of oocyte cumulus complexes. While complete oocyte in vitro development has been achieved in mouse, with the production of live offspring, the goal of obtaining oocytes of sufficient quality to support embryo development has not been completely reached into higher mammals despite decades of effort. CONCLUSIONS: Over the years, many improvements have been made on ovarian tissue cultures with the future purpose that patients will be provided with a greater number of developmentally competent oocytes for fertility preservation.

ABSTRACT: Aim: To evaluate if anti-Müllerian hormone (AMH) is a reliable marker of ovarian reserve in young women undergoing ovarian tissue cryopreservation. Patients & methods: Relationships of serum AMH levels with primordial follicle density, age and reproductive hormones were investigated using the Pearson or Spearman correlation coefficient in 86 women with cancer (12–38 years) undergoing ovarian tissue cryopreservation. AMH variations through the menstrual cycle were assessed by the Kruskal–Wallis test. p

ABSTRACT: 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.

Objective To investigate the ovarian reserve in female lymphoma patients and the potential relationships with the cytokine network. Design Age-matched control study. Setting Women's 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 antimullerian 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.

Aim: To investigate mechanisms by which doxorubicin (DOX) and cisplatin (CIS) cause human ovarian stroma injury. Patients & methods: Stromal cells from human cryopreserved ovarian tissue were cultured in the presence of 1 µM DOX and 10 µM CIS. Ovarian damage induced by treatments was evaluated by ‘Live/Dead’ and sulforhodamine-B assays, the expression of different apoptosis markers. Results: Stromal cell growth was inhibited by DOX and CIS, and this effect was accompanied by apoptosis through mitochondrial pathway activation: Bax, cleaved-caspase 9, cleaved-PARP1 induction and Akt1, Bcl2, phospho-44/42-MAPK/ERK1/2 reduction were observed. Conclusion: DOX and CIS induced apoptosis in human ovarian stromal cells. Knowledge of mechanisms by which the drugs act is important to identify possible ways to counteract side effects of chemotherapy on ovaries.

Ovarian tissue cryopreservation (OTC), representing a promising strategy to preserve ovarian function in cancer patients, is recommended to women younger than 35 years. This study aimed to identify endocrine and biometric parameters as additional selection criteria for OTC. One hundred and ninety-one cancer patients before chemoradiotherapy and OTC and 43 controls were investigated. Mean ± SD, median, quartiles, 5th and 95th centiles and correlations of FSH, LH, estradiol, inhibin-B, anti-Mullerian hormone (AMH), ovarian volume and antral follicle count (AFC) were assessed. Most ovarian reserve parameters presented typical variations of ovulatory menstrual cycle, except AMH and AFC showing minimal fluctuations across the menstrual cycle. The 5th centiles of AMH (0.31and 0.4 ng/mL in controls and cancer patients, respectively) and AFC (five follicular structures in both groups) could be conjectured as minimum thresholds to include patients aged

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.

Study question Which is the best method for human ovarian tissue cryopreservation: slow freezing/rapid thawing (SF/RT) or vitrification/warming (V/W)? Summary answer The conventional SF/RT protocol used in this study seems to better preserve the morpho-functional status of human cryopreserved ovarian tissue than the used open carrier V/W protocol. What is known already Cryopreservation of human ovarian tissue is generally performed using the SF/RT method. However, reduction in the follicular pool and stroma damage are often observed. An emerging alternative procedure is represented by V/W which seems to allow the maintenance of the morphological integrity of the stroma. Study design, size, duration This is a retrospective cohort study including six patients affected by oncological diseases and enrolled from January to December 2014. Participants/materials, setting, methods Ovarian tissue was laparoscopically harvested from the right and left ovaries and was cryopreserved using a routinary SF/RT protocol or a V/W method, involving tissue incubation in two solutions (containing propylene glycol, ethylene glycol and sucrose at different concentrations) and vitrification in an open system. For each patient, three pieces from each ovary were collected at the time of laparoscopy (fresh tissue) and after storage (SF/RT or V/W) and processed for light microscopy (LM) and transmission electron microscopy (TEM), to assess the morphological and ultrastructural features of follicles and stroma, and for laser scanning confocal microscopy (LSCM), to determine the functional energetic/redox stroma status. The preservation status of SF/RT and V/W ovarian tissues was compared with that of fresh ones, as well as between them. Main results and the role of chance By LM and TEM, SF/RT and V/W samples showed cryodamage of small entity. Interstitial oedema and increased stromal cell vacuolization and chromatin clumping were observed in SF/RT samples; in contrast, V/W samples showed oocyte nuclei with slightly thickened chromatin and irregular shapes. The functional imaging analysis by LSCM revealed that the mitochondrial activity and intracellular reactive oxygen species levels were reduced both in SF/RT and in V/W samples compared with fresh samples. The study also showed progressive dysfunction of the mitochondrial activity going from the outer to the inner serial section of the ovarian cortex. The reduction of mitochondrial activity of V/W samples compared with fresh samples was significantly higher in the inner section than in the outer section. Limitations, reasons for caution The results report the bioenergetic and oxidative status assessment of fresh and cryopreserved human ovarian tissue by LSCM, a technique recently applied to tissue samples. The use of LSCM on human ovarian tissues after SF/RT or V/W is a new application that requires validation. The procedures for mitochondrial staining with functional probes and fixing are not yet standardized. Xenografting of the cryopreserved ovarian tissue in severe combined immunodeficient mice and in vitro culture have not yet been performed. Wider implications of the findings The identification of a cryopreservation method able to maintain the morpho-functional integrity of the ovarian tissue and a number of follicles comparable with those observed in fresh tissue might optimize results in clinical practice, in terms of recovery, duration of ovarian function and increased delivery outcomes after replanting. The SF/RT protocol allowed better morpho-functional tissue integrity than the V/W procedure. Study funding/competing interests Funding was provided by Fondazione del Monte di Bologna e Ravenna, Italy. Dr N.A.M. was granted by the project ONEV MIUR PONa3 00134-n.254/R&C 18 5 2011 and the project GR-2011-02351396 (Ministry of Health, Young Researchers Grant 2011/2012). There are no competing interests. Trial registration number Clinical trial 74/2001/0 (approved:13 2 2002): 'Pilot study on cryopreservation of human ovarian tissue: morphological and immunohistochemical analysis before and after cryopreservation'.

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.

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.

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 . 0 0 0 1 ). 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.

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).

Tendons transmit forces generated from muscle to bone making joint movements possible. Tendon collagen has a complex supramolecular structure forming many hierarchical levels of association; its main functional unit is the collagen fibril forming fibers and fascicles. Since tendons are enclosed by loose connective sheaths in continuity with muscle sheaths, it is likely that tendon sheaths could play a role in absorbing/transmitting the forces created by muscle contraction. In this study rat Achilles tendons were passively stretched in vivo to be observed at polarized light microscope (PLM), scanning electron microscope (SEM) and transmission electron microscope (TEM). At PLM tendon collagen fibers in relaxed rat Achilles tendons ran straight and parallel, showing a periodic crimp pattern. Similarly tendon sheaths showed apparent crimps. At higher magnification SEM and TEM revealed that in each tendon crimp large and heterogeneous collagen fibrils running straight and parallel suddenly changed their direction undergoing localized and variable modifications. These fibril modifications were named fibrillar crimps. Tendon sheaths displayed small and uniform fibrils running parallel with a wavy course without any ultrastructural aspects of crimp. Since in passively stretched Achilles tendons fibrillar crimps were still observed, it is likely that during the tendon stretching, and presumably during the tendon elongation in muscle contraction, the fibrillar crimp may be the real structural component of the tendon crimp acting as shock absorber. The peritendinous sheath can be stretched as tendon, but is not actively involved in the mechanism of shock absorber as the fibrillar crimp. The different functional behaviour of tendons and sheaths may be due to the different structural and molecular arrangement of their fibrils.

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).