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STUDY QUESTION: Does vitrification of human immature testicular tissue (ITT) have potential benefits for future fertility preservation? Does vitrification of human ITT have potential benefits in an in vivo murine xenotransplantation model? SUMMARY ANSWER: Vitrification is able to maintain proliferation capacity in spermatogonial cells after 6 months of xenografting. WHAT IS KNOWN ALREADY: Controlled slow-freezing is the procedure currently applied for ITT cryobanking in clinical practice. Vitrification has been proposed as a promising technique for long-term storage of ITT, with a view to preserving spermatogonial stem cells (SSCs) for future fertility restoration in young boys suffering from cancer. After vitrification of ITT, in vitro survival of SSCs was demonstrated, but their functionality was not evaluated. STUDY DESIGN, SIZE, DURATION: Ten ITT pieces issuing from 10 patients aged 2-12 years were used. Fragments of fresh tissue (serving as controls) and fresh, frozen-thawed and vitrified-warmed testicular pieces xenografted to the scrotum of nude mice for 6 months were compared. MATERIALS, SETTING, METHODS: Upon graft removal, histological and immunohistochemical analyses were performed to evaluate spermatogonia (SG) (MAGE-A4), intratubular proliferation (Ki67), proliferating SG and Leydig cells (3β-HSD). The entire piece of grafted tissue was assessed in each case. MAIN RESULTS AND THE ROLE OF CHANCE: Seminiferous tubules showed good integrity after cryopreservation and xenografting for 6 months in all three groups. Survival of SG and their ability to proliferate was observed by immunohistochemistry in all grafted groups. SG were able to initiate spermatogenesis, but blockage at the pachytene stage was observed. The recovery rate of SG was 3.4 ± 3.8, 4.1 ± 7.3 and 7.3 ± 6.3%, respectively, for fresh, slow-frozen and vitrified-warmed tissue after 6 months of xenografting. LIMITATIONS, REASONS FOR CAUTION: The study is limited by the low availability of ITT samples of human origin. The mouse xenotransplantation model needs to be refined to study human spermatogenesis. WIDER IMPLICATIONS OF THE FINDINGS: The findings of the present study have potential implications for cryobanking of ITT and fertility preservation. Spermatogonial loss recorded after fresh ITT transplantation indicates that the avascular grafting technique needs to be optimized. There are so far no convincing data justifying modification of current clinical practice for ITT storage with slow-freezing, but this study demonstrates that it is worth pursuing optimization of ITT vitrification as an alternative for preservation of SSCs. STUDY FUNDING/COMPETING INTEREST(S): The present study was supported by a grant from the Fonds National de la Recherche Scientifique de Belgique (grant Televie N(°) 7. 4.572.09.F). The authors declare that there is no conflict of interest.

Objective To identify metabolites that are associated with and predict the presence of endometriosis. Design Metabolomics study using state-of-the-art mass spectrometry approaches. Setting University hospital and universities. Patient(s) Twenty-five women with laparoscopically confirmed endometriosis (cases) and 19 women with laparoscopically documented absence of endometriosis (controls). None of the women included in this study had received oral contraception or GnRH agonists for a minimum of 1 month before blood collection. Intervention(s) Plasma collection. Main Outcome Measure(s) Metabolite profiles were generated and interrogated using multiple mass spectrometry methods, that is, high performance liquid chromatography coupled with negative mode electrospray ionization tandem mass spectrometry, UPLC-MS/MS, and ultra performance liquid chromatography-electroSpray ionization-quadrupole time-of-flight (UPLC-ESI-Q-TOF). Metabolite groups investigated included phospholipids, glycerophospholipids, ether-phospholipids, cholesterol-esters, triacylglycerol, sphingolipids, free fatty acids, steroids, eicosanoids, and acylcarnitines. Result(s) A panel of acylcarnitines predicted the presence of endometriosis with 88.9% specificity and 81.5% sensitivity in human plasma, with a positive predictive value of 75%. However, due to data limitations the outcome of the receiver operating characteristic curve analysis was not significant. Conclusion(s) A diagnostic model based on acylcarnitines has the potential to predict the presence and stage of endometriosis.

The efficiency of oocyte in-vitro maturation (IVM) and vitrification procedures after ex-vivo collection from ovarian tissue were assessed according to patient age, number of retrieved oocytes and tissue transport conditions. The combined procedure was performed in 136 patients: 130 adults (mean 27.6 ± 5.6 years) and six prepubertal girls (mean 8.7 ± 2.3 years). A higher mean number of oocytes were collected in girls compared with adults (11.5 ± 8.0 versus 3.8 ± 4.2, respectively, P < 0.001) but the percentage of degenerated oocytes was significantly higher in girls (35.5% versus 17.1%, respectively, P < 0.001). IVM rates were significantly lower in prepubertal than postpubertal population (10.3% versus 28.1%, P = 0.002). In adults, a negative correlation was observed between number of retrieved oocytes and age (P = 0.002; r = -0.271); the correlation was positive between anti-Mullerian hormone (AMH) and number of collected oocytes (P = 0.002; r = 0.264). IVM rates were not correlated with AMH levels (r = 0.06) or age (r = -0.033). At present, nine oocytes and one embryo have been warmed in four patients and one biochemical pregnancy obtained. This suggests the combined procedure could be an additional option for fertility preservation.

BACKGROUNDAlthough cryopreservation and transplantation of ovarian tissue represent a promising alternative to safeguard fertility in cancer patients, low recovery rates of oocytes aspirated from antral follicles and a significant number of empty follicles have been observed in women with transplanted frozen-thawed ovarian tissue. In order to understand how freezing and/or grafting may affect follicular development, the follicular expression of kit ligand (KL) and anti-Mullerian hormone (AMH), two key factors activating and inhibiting follicle growth, were assessed after long-term grafting in severe combined immunodeficient (SCID) mice.METHODSOvarian biopsies from eight patients were used for fresh and frozen-thawed tissue xenografting in 13 SCID mice for a period of 28 weeks, including 2 weeks of gonadotrophin stimulation. KL, AMH and proliferating cell nuclear antigen (PCNA) immunostaining were quantified before and after grafting in the two treatment groups (fresh and frozen-thawed grafted ovarian tissue).RESULTSLower expression of KL was found in primordial and primary follicles after grafting of both fresh and frozen-thawed tissue. Consistent expression of AMH was found in most growing follicles at a similar rate in both graft types. In fresh and frozen-thawed grafts, 13-14% of primordial follicles were PCNA-positive, indicating a similar maintenance of quiescent follicles despite follicle activation.CONCLUSIONSGrafting and/or gonadotrophin stimulation appear to affect the follicular expression of KL, which may alter oocyte quality. AMH expression in growing follicles after ovarian tissue transplantation may be one of the factors contributing to the preservation of resting follicles in 28-week-old grafts.

In this study, we developed an original and reproducible quantitative in vitro model of endometrial cell adhesion to peritoneal mesothelial cells in order to better assess the impact of pro-inflammatory cytokines on early-stage endometriosis development. We demonstrated that pre-treatment with TNF-α and IL-1β does not promote endometrial stromal cell adhesion to peritoneal mesothelial cells.

OBJECTIVE: To test the effect of different vitrification solutions and procedures on the morphology of human preantral follicles. DESIGN: Pilot study. SETTING: Gynecology research unit in a university hospital. PATIENT(S): Ovarian biopsies were obtained from nine women aged 22-35 years. INTERVENTION(S): Ovarian tissue fragments were subjected to [1] different vitrification solutions to test their toxicity or [2] different vitrification methods using plastic straws, medium droplets, or solid-surface vitrification before in vitro culture. MAIN OUTCOME MEASURE(S): Number of morphologically normal follicles after toxicity testing or vitrification with the different treatments determined by histologic analysis. RESULT(S): In the toxicity tests, only VS3 showed similar results to fresh tissue before and after in vitro culture (fresh controls 1 and 2). In addition, this was the only solution able to completely vitrify. In all vitrification procedures, the percentage of normal follicles was lower than in controls. However, of the three protocols, the droplet method yielded a significantly higher proportion of normal follicles. CONCLUSION(S): Our experiments showed VS3 to have no deleterious effect on follicular morphology and to be able to completely vitrify, although vitrification procedures were found to affect human follicles. Nevertheless, the droplet method resulted in a higher percentage of morphologically normal follicles.

OBJECTIVE: To characterize the human ovarian xenograft revascularization process. DESIGN: Prospective experimental study. SETTING: Gynecology research unit in a university hospital. PATIENT(S): Ovarian biopsies were obtained from 12 women aged 22-35 years. INTERVENTION(S): Frozen-thawed human ovarian fragments were intraperitoneally grafted into nude mice. MAIN OUTCOME MEASURE(S): Graft perfusion was evaluated by Hoechst 33342 uptake. Murine and human vascularization was analyzed by CD31 and von Willebrand factor double immunostaining. RESULT(S): On day 3, some murine neovessels and perfused areas were located at the periphery of the fragments. Nonperfused native human vessels were present in the fragments. From day 5, perfused areas and murine endothelial areas progressively increased. Host angiogenesis initiated ovarian graft reperfusion: murine neovessels penetrated from the periphery and were colocalized with perfused areas. By day 10, the increase in perfusion and murine vascularization was significant. The center of the fragments was perfused and a significant increase was observed in human vasculature. CONCLUSION(S): Host and graft vessels contributed sequentially to graft revascularization: murine angiogenesis initiated reperfusion from day 5 and, by day 10, human angiogenesis was shown to participate in graft revascularization. Host and graft angiogenesis are potential targets to reduce the avascular period after grafting.

Fertility in adult life may be severely impaired by gonadotoxic therapies. For young boys who do not yet produce spermatozoa, cryopreservation of immature testicular tissue (ITT) is an option to preserve their fertility, albeit still experimental. This paper covers current options for ITT cryopreservation and fertility restoration. Relevant studies were identified by an extensive Medline search of English and French language articles. Search terms were: gonadotoxicity, cytoprotection, cryopreservation, ITT, spermatogonia, testicular transplantation, testicular grafting and in vitro maturation (IVM). Although no effective gonadoprotective drug is yet available for in vivo spermatogonial stem cell protection in humans, current evidence supports the feasibility of ITT cryopreservation before gonadotoxic treatment with a view to fertility preservation. Controlled slow freezing with dimethyl sulfoxide allows survival and proliferation of human spermatogonia after xenotransplantation, but only partial differentiation. Animal data look promising, since healthy offsprings have been obtained after transplantation of frozen testicular cell suspensions or tissue pieces. However, none of the fertility restoration options from frozen tissue, i.e. cell suspension transplantation, tissue grafting and IVM have proved efficient and safe in humans as yet. While additional evidence is required to define optimal conditions for ITT cryopreservation with a view to transplantation or IVM, the putative indications for such techniques, as well as their limitations according to disease, are outlined.

Classic murine endometriosis models may be insufficient to evaluate the effect of therapeutic agents on endometriosis development, because the process of identification and measurement of induced lesions is often impeded, as implants are small and embedded in murine tissue. In this context, as summarized in the current review, luminescence techniques have proved useful for identifying and visualizing or quantifying endometriotic transplants. They are also a valuable tool for endometrial cell tracking in live animals, yielding further information by adding spatial and temporal dimensions to biological processes in vivo. Such approaches involve transplanting luminescently labeled murine or human endometrium into animals. Two main strategies are applied to label endometrium before injection: use of genetically modified tissue or tissue labeled with a fluorescent dye. Each model has its advantages and disadvantages, the choice of model depends on the study objectives/design (long- or short-term studies, homologous or heterologous model).

BACKGROUND Chemo- or radiotherapy can induce premature ovarian failure (POF), and ovarian tissue cryopreservation and transplantation may be proposed to restore ovarian function. Our aim was to evaluate the quality of oocytes and embryos derived from frozen-thawed transplanted ovarian tissue. MATERIALS AND METHODS Women were 21-28 years old at tissue cryopreservation. Nine women suffering POF following chemotherapy with or without radiotherapy underwent orthotopic ovarian tissue transplantation. After 12 months of spontaneous cycles without pregnancy, oocyte retrieval was performed in four patients during mildly stimulated or spontaneous cycles. ICSI was performed in all cases, with embryo transfer on day 3. Light and electron microscopy was used to study oocytes and embryos. RESULTS Signs of ovarian function restoration (estradiol peak, decreased FSH, follicular development) began 16-26 weeks after reimplantation. Twenty-one oocyte retrieval attempts were made. At least one oocyte was collected in 15 cases, giving an empty follicle rate per retrieval of 29% (6/21). Sixteen oocytes were recovered, of which 6 were abnormal or immature (38%) and 10 (62%) were in metaphase II (MII). Three MII oocytes failed to fertilize, two showed abnormal fertilization and five normal MII oocytes successfully fertilized with subsequent normal embryo development (Grade 2), yielding an embryo transfer rate of 24% per retrieval. No pregnancy occurred. CONCLUSIONS IVF in women with orthotopically grafted frozen-thawed ovarian tissue involves a higher risk of empty follicles, abnormal or immature oocytes, and low embryo transfer rates.

OBJECTIVE: To develop electron paramagnetic resonance (EPR) oximetry as a tool to characterize the oxygen environment in human ovarian xenografts in the early postgrafting period. DESIGN: Prospective experimental study. SETTING: Gynecology research unit in a university hospital. PATIENT(S): Biopsies were obtained from 6 women aged 22-35 years. INTERVENTION(S): Frozen-thawed human ovarian tissue fragments were grafted to an intraperitoneal site in nude mice. Before grafting, lithium phthalocyanine, an oxygen reporter, was implanted inside the fragments. MAIN OUTCOME MEASURE(S): To monitor partial pressure of oxygen (pO(2)) by EPR on postgrafting days 3, 5, 7, 10, 14, 17, and 21 and validate the technique by histologic assessment. RESULT(S): A period of hypoxia was identified before day 5, followed by gradual but significant oxygenation over the next 5 days, suggesting an active process of graft revascularization. Reoxygenation kinetics in human ovarian xenotransplants were quantified. CONCLUSION(S): Our data validated the EPR oximetry technique as a tool to monitor pO(2) in ovarian grafting. The critical early period of hypoxia was identified, and the first steps of reoxygenation were characterized. In the future, our model may be used to evaluate new freezing and grafting protocols with the aim of reducing potential cryoinjury and initial ischemia-reperfusion damage.

Purpose Ectopic endometrial tissue is biochemically and functionally distinct from eutopic endometrium, including differences in receptivity to steroids and invasive potential. For this reason, using ectopic endometrial tissue to induce endometriosis in animal models may be of interest to understand better the pathogenesis of this disease. The aim of this study was to evaluate the potential of human pelvic endometriotic lesions (specifically red lesions) to implant into the peritoneal cavity of nude mice. Methods Human peritoneal endometriotic tissue (red lesions) was injected into the pelvic cavity of nude mice. The mice were euthanized after 5 or 14 days, lesions were recovered, and histological analysis was performed. Proliferative activity and neoangiogenesis of lesions were assessed after Ki67 and CD34 immunohistological staining, respectively. Results After 5 and 14 days, endometriotic tissue was found to have implanted onto intact mesothelium and reorganized into structured glands and stroma, forming endometriosis-like lesions. These lesions showed considerable proliferative activity and effective neoangiogenesis. Conclusion This model represents a promising tool to study in vivo implantation of human ectopic endometrium, which is dissimilar to eutopic endometrium. Moreover, this study shows that glands and stroma detached from red endometriotic lesions are able to implant in other peritoneal sites, as in a metastatic process.

Peritoneal endometriosis is a chronic pelvic inflammatory disease, characterized by increased numbers of peritoneal macrophages and their secreted products such as cytokines, growth and angiogenic factors in peritoneal fluid. Inflammation plays a major role in pain and infertility associated with endometriosis, but is also extensively involved in the molecular and cellular processes that lead to peritoneal endometriotic lesion development. Several inflammatory mediators have therefore been studied in the context of endometriosis over the last few years. The aim of this review is to focus on three that have been clearly implicated in the pathogenesis of endometriosis and may be linked: peritoneal iron metabolism, nuclear factor-kappa B (NF-κB) activation, and prostaglandin biosynthesis. Peritoneal iron overload has been conclusively demonstrated in endometriosis patients and may induce oxidative stress in the peritoneal cavity. Oxidative stress and proinflammatory cytokines are well known to be potent activators of the NF-κB pathway, which has recently been implicated in peritoneal endometriosis. Induced NF-κB activation leads to expression of numerous proinflammatory genes such as cytokines, which may provide positive feedback to the pathway, self-perpetuating the inflammatory response. Other important NF-κB-regulated molecules are prostaglandin biosynthesis enzymes, and cyclooxygenase-2 (COX-2) in particular. Increased concentrations of prostaglandins have been evidenced in the peritoneal fluid of endometriosis patients and COX-2 inhibitors have proved to be effective in ‘in vitro’ and ‘in vivo’ experimental models. In the light of available data collected from patient biopsies, as well as ‘in vitro’ and ‘in vivo’ studies, the respective implication and potential molecular association of iron, NF-κB and prostaglandins in the pathogenesis of endometriosis are discussed. The key role of peritoneal macrophages is emphasized and potential therapeutic targets are examined.

Background/Aims: Endometriosis is known to be an estrogen-dependent disease. However, only a few studies have analyzed the effect of estrogen treatment in mice xenotransplanted with human endometrium. The objective of this study was to adapt a previously developed heterologous murine model to the study of estrogens and test the impact of estrone treatment on endometriosis development. Methods: Human proliferative endometrium was xenotransplanted into the peritoneal cavity of castrated immunodeficient mice. These mice were treated with estrogens by means of subcutaneous estrone-releasing pellets. The effect of estrone on estradiol level, uterine histology and endometriosis development was evaluated after 21 days. Results: Bioactivity of estrone pellets and their metabolization into estradiol were demonstrated. However, there was no impact on endometriosis development (no difference in lesion number, weight, size or fluorescence). This lack of response was not due to absence of estrogen receptor expression, since strong expression was found in all lesions harvested. Surprisingly, castrated nontreated mice presented with lesions showing high proliferative activity, similar to lesions found in treated mice (around 30%). Conclusion: The high proliferation observed in lesions recovered from ovariectomized nontreated mice questions the utility of using estrogens in heterologous murine models.

This is the first report of the presence of ultrastructurally normal primordial and primary follicles, 13 months after autotransplantation of frozen-thawed human ovarian tissue. The stroma contained numerous viable and ultrastructurally normal blood vessels but showed poor cellular density.

It is widely known that angiogenesis plays a key role in endometriotic lesion formation and development. Antiangiogenic treatments aimed at inhibiting new vessel formation have proven efficient in experimental models. However, as antiangiogenic strategies do not target pre-existing pericyte-protected vessels, they require chronic administration and are likely to be beneficial for early-stage disease only or to prevent recurrence after surgery. Moreover, they may have detrimental effects on reproductive function. Vascular-disrupting agents (VDAs) have emerged as a promising new tool for the treatment of tumors. VDAs target established blood vessels, resulting in tumor ischemia and necrosis. These agents may therefore be more efficient against advanced disease. Two major types of VDAs are being developed for cancer: ligand-directed VDAs using antibodies, peptides and growth factors to deliver toxic effectors to tumor endothelium; and small-molecule VDAs exploiting physiological differences between tumor and normal endothelium to induce acute vascular shutdown. The ongoing evolution in genomics and proteomics is revolutionizing the discovery of novel endothelial markers. Several studies suggest that the vasculature of endometriotic lesions may have particular pathophysiological properties, which could be exploited for the development of selective VDAs. The aim of this review is to explore the merits and limitations of vascular therapy for the treatment of endometriosis.

Cryopreservation of ovarian tissue is currently proposed to young cancer patients before chemo- or radiotherapy to preserve their fertility. In this study, ovarian cortex was removed by laparoscopy from five women and cryopreserved before chemotherapy. After chemotherapy, they all experienced amenorrhoea due to premature ovarian failure and requested reimplantation of their cryopreserved ovarian tissue several years later. Thawed fragments were then grafted to an orthotopic site in all five women. Two of them underwent a second reimplantation. Ovarian function recovery was evaluated by hormone concentration measurement, follicular development on ultrasound and menstruation recovery. The first signs of ovarian function restoration (oestradiol peak, decrease in FSH, ultrasound showing follicular development) occurred between 16 and 26 weeks after reimplantation. Elevated FSH concentrations were sometimes observed between series of consecutive ovulatory cycles, demonstrating the presence of a relatively low ovarian reserve. There were no signs of disease recurrence in any patients with malignant disease. In conclusion, restoration of ovarian function was observed in all cases. Grafts remained functional in all the women. Transplantation of cryopreserved ovarian tissue to an orthotopic site appears to restore ovarian endocrine function, without any signs of disease recurrence.

Background/Aims: The aim of this study was to induce endometriosis in female rhesus macaques (Macaca mulatta) for research purposes. Methods: Three female monkeys from 4 to 4.5 years of age underwent three consecutive attempts at endometriosis induction over an 8-month period: (i) the first attempt involved intravaginal sampling of endometrial tissue and transplantation into the intrapelvic cavity; (ii) the second entailed surgical removal of endometrium after hysterotomy and intra-abdominal placement, and (iii) the third used endometrial mucosa obtained by scraping the uterus after hysterectomy, placed in a surgical pouch created in the retrovesical space (Retzius). In each case, the pelvic cavity was closely inspected after 7, 9, and 6 weeks respectively for the presence of endometriotic lesions, and peritoneal biopsies were performed. Results: Neither macroscopic observation nor histological analysis revealed any endometriotic lesions. Conclusion: This failure to induce endometriosis in female rhesus macaques suggests that this species is not the most efficient experimental model among primates to investigate endometriosis development or treatment.

Background/Aims: Endometrial cells are chronically exposed to iron due to cyclic menstrual bleeding. Iron induces expression of adhesion molecules in endothelial cells. The purpose of this study was to investigate iron incorporation by human endometrial cells and to test whether iron may stimulate expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1. Methods: Endometrial stromal and epithelial cells were cultured in medium alone or supplemented with INF-γ or transferrin (Tf). Iron incorporation by cells was quantified by densitometry of ferritin immunostaining. ICAM-1 and VCAM-1 expression were evaluated at the transcriptional level by real-time RT-PCR. Membrane-bound and soluble protein levels of ICAM-1 were measured by quantitative immunohistochemistry and ELISA, respectively. Results: Tf induced a significant increase in ferritin immunostaining in both endometrial cell types. Endometrial cells treated with INF-γ expressed more ICAM-1 and VCAM-1 than untreated cells. By contrast, Tf treatment did not alter ICAM-1 and VCAM-1 expression in cultured endometrial cells. Conclusions: Endometrial cells are able to incorporate iron from Tf and to metabolize it to ferritin. Iron, unlike interferon-γ, does not appear to be involved in the regulation of ICAM-1 and VCAM-1 expression in cultured endometrial cells.

This study was designed to evaluate follicular survival and growth after short-term transplantation of fresh isolated human follicles and ovarian cortical tissue to nude mice. Ovarian biopsies were obtained from nine women undergoing laparoscopy. Twelve nude mice were xenografted with an ovarian cortical fragment in the right ovarian bursa, and a clot containing isolated follicles in the left, for a period of 7 days. One ungrafted fragment was used as a control. Histological sections were analyzed to determine follicle number and stage. The proliferative status of follicular cells was assessed by Ki-67 immunostaining. A total of 659 follicles was analyzed by histology and 545 follicles by immunohistochemistry. The percentage of primordial follicles was found to be markedly reduced 1 week post-grafting when compared with ungrafted tissue, while the percentage of primary follicles had significantly increased. Only 8% of follicles showed Ki-67-positive granulosa cells before grafting, whereas 1 week after grafting, 71% of follicles in fragments and 67% of isolated follicles were Ki-67-positive (Pin situ hybridization with human-specific probes. Our results demonstrate, for the first time, that isolated human follicles are able to survive and grow after xenografting. This study also shows massive in vivo follicular activation after transplantation of grafted fragments and isolated follicles. One week after grafting, well-structured stroma-like tissue of human origin was observed around the isolated follicles. The potential origin of this stroma is discussed.

OBJECTIVE: To investigate possible damage caused by freeze-thawing whole human ovaries. DESIGN: Prospective experimental study. SETTING: Academic gynecology research unit in a university hospital. PATIENT(S): Ovaries were obtained from three women (aged 29-36 years). INTERVENTION(S): Ovaries were perfused and bathed in cryoprotective solution, and slow freezing was performed. Rapid thawing was achieved by perfusion and bathing with a decreased sucrose gradient. MAIN OUTCOME MEASURE(S): Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphates nick end-labeling (TUNEL) method and by immunohistochemistry for active caspase-3 in fresh ovaries, after cryoprotectant exposure, and after thawing. Morphometric analysis of TUNEL-positive surface area was performed. Ultrastructure was assessed by transmission electron microscopy (TEM) in the thawed tissue. RESULT(S): No primordial or primary follicles were found to be positive for either TUNEL or active caspase-3. No statistically significant difference in mean TUNEL-positive surface area values was found between the three groups: fresh, 0.05% +/- 0.03%, with 134 high-power fields (HPFs); cryoperfused, 0.02% +/- 0.01%, with 130 HPFs; and thawed, 0.09% +/- 0.03%, with 622 HPFs. By means of TEM, follicles and vessels showed a well-preserved ultrastructure, with 96.7% (29/30) healthy-looking primordial and primary follicles, and 96.3% (180/187) healthy-looking endothelial cells. CONCLUSION(S): Cryopreservation of intact human ovary with its vascular pedicle, according to the freeze-thawing protocol described here, is not associated with any signs of apoptosis or ultrastructural alterations in any cell types. Whole-organ vascular transplantation may thus be a viable option in the future.

The review covers current options for ovarian tissue cryopreservation and transplantation and provides a systematic review of the existing literature from the last 10 years, taking into account all previously published reviews on the subject. The different cryopreservation options available for fertility preservation in cancer patients are embryo cryopreservation, oocyte cryopreservation and ovarian tissue cryopreservation. The choice depends on various parameters: the type and timing of chemotherapy, the type of cancer, the patient's age and the partner status. The different options and their results are discussed, as well as their putative indications and efficacy. The review concludes that advances in reproductive technology have made fertility preservation techniques a real possibility for patients whose gonadal function is threatened by premature menopause, or by treatments such as radiotherapy, chemotherapy or surgical castration.

OBJECTIVE: To assess the timing of endometrial lesion revascularization in a murine model. DESIGN: Prospective experimental study. SETTING: An academic research environment. ANIMAL(S): Twenty-six nude mice. INTERVENTION(S): Endometriosis was induced in mice by intraperitoneal deposition of human menstrual endometrium. Endometrial implants were recovered on days 1, 3, 5, 8, 10, 15, and 21 after implantation. MAIN OUTCOME MEASURE(S): Sections from the endometrial implants were immunostained with species-specific antiplatelet endothelial cell adhesion molecule-1 (PECAM-1) antibodies and vessels of murine and human origin were counted. RESULT(S): Endothelial cells of human origin in the implant progressively disappeared between day 3 and day 10. Seventy-eight percent of the vessel sections were positive for human PECAM-1 on day 5, 40.1% on day 8, and only 14.1% on day 10. However, there was a marked increase in murine PECAM-1-expressing vessels in endometrial stroma between day 5 (1.4%) and day 8 (68.0%), 10 (69.5%), and 15 (87.2%). CONCLUSION(S): Our study demonstrates that PECAM-1 is a reliable endothelial cell marker to evaluate the role of angiogenesis in the nude mouse model. It also indicates that revascularization of human endometrial implants occurs between 5 and 8 days after implantation and involves the disappearance of native graft vessels, coinciding with the invasion of the interface and then the stroma by murine vessels.

PROBLEM: Intercellular adhesion molecule-1 (ICAM-1) is thought to play an important role in pathophysiological processes in endometrial tissue. The aim of this study was to quantify and compare the expression of ICAM-1 mRNA and protein in cultured endometrial epithelial cells (EEC) versus endometrial stromal cells (ESC). METHOD OF STUDY: EEC and ESC were isolated from human endometrium and cultured. At confluency, ICAM-1 mRNA was measured by real-time reverse transcriptase-polymerase chain reaction, the membrane-bound form (mICAM-1) by immunocytodensitometry, and the soluble form (sICAM-1) by enzyme-linked immunosorbent assay. RESULTS: At the transcriptional level, we observed a 1.7-fold increase in ICAM-1 expression in EEC versus ESC. mICAM-1 immunostaining in EEC [cell-relative membrane-bound signal-specific optical density (CR-M-SOD): 0.056 +/- 0.05] was stronger (P < 0.05) than in ESC (CR-M-SOD: 0.009 +/- 0.07). EEC were found to secrete more (P < 0.01) sICAM-1 ([sICAM-1] = 15.59 +/- 2.96 ng/mL) than ESC ([sICAM-1] = 5.14 +/- 2.61 ng/mL). CONCLUSIONS: Cultured EEC constitutively express significantly more ICAM-1 mRNA and protein than ESC.

OBJECTIVE: To develop a simple and efficient technique to allow rapid recovery of a maximum number of good quality isolated follicles. DESIGN: Prospective experimental study. SETTING: Academic research unit of the department of gynecology in a university hospital. PATIENT(S): Biopsies were obtained from five women (between 26 and 31 years of age). INTERVENTION(S): Biopsies were cut with a tissue sectioner. Enzymatic digestion was performed in a collagenase solution for 90 min at 37 degrees C. The follicles were recovered using a discontinuous Ficoll density gradient method. MAIN OUTCOME MEASURE(S): The number of follicles present in the interface layers of Ficoll gradient was quantified. Follicular viability of these recovered follicles was assessed with live-dead stains, using calcein-AM and ethidium homodimer-I. RESULT(S): Out of a total of 6,811 recovered follicles, we found 63% (n = 4,201) at the medium-1.06 Ficoll interface and 36.9% (n = 2,590) at the 1.06-1.09 Ficoll interface, which represents 99.9% of total recovered follicles. Analysis by vital fluorescent staining showed that 95.8% of the follicles treated with Ficoll were totally viable. CONCLUSION(S): The Ficoll density gradient method allows us to maximize the recovery of isolated human ovarian follicles and minimize the manipulation time while maintaining high follicular viability.

BACKGROUND: Pelvic endometriotic lesions are often laden with hemosiderin. In order to investigate the potential source of such iron deposits, we examined whether the seric and erythrocyte fractions of menstrual effluent might influence the occurrence of iron deposition within lesions in a murine model of endometriosis. METHODS: Endometriosis was induced in 57 nude mice by intraperitoneal injection of unfractionated human menstrual effluent, endometrial fragments plus serum, endometrial fragments plus erythrocytes or endometrial cell fraction alone. The number of implants, histologic aspect, proliferative activity and iron deposition in lesions was assessed. RESULTS: On day 5, lesions were evidenced in all 10 mice receiving menstrual effluent, in 9/13 of those injected with the cellular fraction, in 10/13 with the cellular fraction plus serum and in 10/12 with the cellular fraction plus erythrocytes. Iron conglomerates were observed at the interface between the lesion and peritoneum when menstrual effluent (47 deposits/mm(2)) and the cellular fraction with erythrocytes (20 deposits/mm(2)) were injected, but were scarce when the cellular fraction was injected without erythrocytes, either alone (4 deposits/mm(2)) or with serum (2 deposits/mm(2)) (P < 0.05). CONCLUSIONS: Iron conglomerates, typically found in the stroma of endometriotic lesions, were induced by erythrocytes present in menstrual effluent. This may be one of the factors triggering oxidative damage and chronic inflammation.

OBJECTIVE: To review the literature associating pelvic endometriosis with oxidative stress and to discuss the potential causes and consequences of a pro-oxidant environment in the peritoneal cavity. DESIGN: Literature survey. RESULT(S): Several studies suggest that oxidative stress is a component of the inflammatory reaction associated with endometriosis. Evidence includes the prevention of endometriosis induction in rabbits by the addition of antioxidants, an increase in reactive oxygen species release by macrophages, increased peritoneal levels of oxidized low-density lipoproteins and their by-products, altered expression of endometrial pro-oxidant and antioxidant enzymes, and consumption of peritoneal fluid vitamin E. Retrograde menstruation is likely to carry highly pro-oxidant factors, such as heme and iron, into the peritoneal cavity, as well as apoptotic endometrial cells, which are well-known inducers of oxidative stress. Reactive oxygen species may be involved in endometriosis-associated infertility and may play a role in the regulation of the expression of genes encoding immunoregulators, cytokines and cell adhesion molecules implicated in the pathogenesis of endometriosis. CONCLUSION(S): Better understanding of the mechanisms of reactive oxygen species production and detoxification and further investigation of their effect on the peritoneal environment are essential to obtain new insight into this disease and eventually develop new diagnostic and therapeutic strategies.

This manuscript is a review of new ideas regarding the pathogenesis of peritoneal endometriosis, ovarian endometriosis, and retroperitoneal adenomyosis. Peritoneal endometriosis, the different aspects of which (black, red and white) represent distinctive steps in the evolutionary process, can be explained by the transplantation theory. Red lesions are the most active and most highly vascularized lesions and are considered to be the first stage of peritoneal endometriosis. The retroperitoneal nodule is an adenomyotic nodule whose histopathogenesis is not related to the implantation of regurgitated endometrial cells but to metaplasia of Müllerian remnants located in the rectovaginal septum. Metaplastic changes of Müllerian rests into adenomyotic glands involving the rectovaginal septum and the retroperitoneal space are responsible for the striking proliferation of the smooth muscle, creating an adenomyomatous appearance similar to that of adenomyosis in the endometrium.

Can a vitrification protocol using an ethylene glycol/dimethyl sulphoxide-based solution and a cryopin successfully cryopreserve baboon ovarian tissue?Our results show that baboon ovarian tissue can be successfully cryopreserved with our vitrification protocol.Non-human primates have already been used as an animal model to test vitrification protocols for human ovarian tissue cryopreservation.Ovarian biopsies from five adult baboons were vitrified, warmed and autografted for 5 months.After grafting, follicle survival, growth and function and also the quality of stromal tissue were assessed histologically and by immunohistochemistry. The influence of the vitrification procedure on the cooling rate was evaluated by a computer model.After vitrification, warming and long-term grafting, follicles were able to grow and maintain their function, as illustrated by Ki67, anti-Müllerian hormone (AMH) and growth differentiation factor-9 (GDF-9) immunostaining. Corpora lutea were also observed, evidencing successful ovulation in all the 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.Despite our promising findings, before applying this technique in a clinical setting, we need to validate it by achieving pregnancies.In addition to encouraging results obtained with our vitrification procedure for non-human ovarian tissue, this study also showed, for the first time, expression of AMH and GDF-9 in ovarian follicles.This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (grant Télévie No. 7.4507.10, grant 3.4.590.08 awarded to Marie-Madeleine Dolmans), Fonds Spéciaux de Recherche, Fondation St Luc, Foundation Against Cancer, and Department of Mechanical Engineering at Louisiana State University (support to Ram Devireddy), and donations from Mr Pietro Ferrero, Baron Frère and Viscount Philippe de Spoelberch. None of the authors has any competing interests to declare.

To evaluate, in peritoneal, ovarian, and rectovaginal endometriotic lesions, expression of steroidogenic enzymes involved in the activation and inactivation of estrogens: 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1) and 2 (HSD17B2), estrone sulfotransferase (EST), and steroid sulfatase (STS).Academic gynecology research unit.Retrospective study.Disease-free (n = 41) patients and patients with endometriosis (n = 79) were included for quantitative polymerase chain reaction (q-PCR) (15 disease-free, 33 endometriosis) and immunohistochemistry (26 disease-free, 46 endometriosis) studies.Q-PCR and immunohistochemistry.Evaluation of mRNA and protein expression.Glandular HSD17B1, HSD17B2, and STS protein expression were demonstrated. HSD17B2 mRNA values were higher in the secretory phase of the menstrual cycle in the endometrium of disease-free women, but not in the eutopic endometrium of patients with endometriosis. HSD17B1 mRNA was equally expressed in the various tissues investigated, and EST mRNA was expressed at low levels in the different lesion types. HSD17B2 mRNA expression was decreased in ovarian and rectovaginal endometriosis compared with eutopic endometrium, while STS mRNA was increased in rectovaginal lesions compared with ovarian lesions. Ratios between pro- and antiestrogenic enzymes (STS/EST and HSD17B1/HSD17B2) were more in favor of estrogens in ovarian and rectovaginal endometriosis.In endometriosis development, local activation of estrogens appears to be important. STS and HSD17B1 inhibitors may therefore prove useful to treat the disease.

To review 15 years of activities in ovarian tissue cryobanking from medical database files, including patient indications, histological evaluation and clinical characteristics.Retrospective longitudinal analysis of data from an ovarian tissue bank in an academic hospital. Five hundred and eighty-two patients had their ovarian tissue cryobanked between April 1997 and January 2012. Analysis of cryobanking database: precryopreservation patient characteristics, indications and safety issues, laboratory files and postcryopreservation clinical data.Of the 582 patients who had their ovarian tissue cryopreserved, 106 patients donated for research purposes and 476 patients for fertility preservation and long-term cryopreservation. Clinical data analysis of the 476 patients revealed a mean age at the time of cryopreservation of 23 ± 8.5 years (range: 9 months - 39 years), with 96.2 % of subjects aged ≤35 years (n = 458). Among 391 cases of malignant disease, hematological malignancies (39.9 %, n = 156) and breast cancer (21.7 %, n = 85) were the two main indications. At histology, malignant cells were found in ovarian tissue from leukemia patients (n = 3) and non-Hodgkin's lymphoma patients (n = 2). Eleven patients underwent autotransplantation, resulting in 5 live births and 1 ongoing pregnancy.This is the largest and most comprehensive study to describe and analyze indications and clinical patient characteristics before and after ovarian tissue cryopreservation. The procedure is safe, easy and promising. The database concept is a useful tool in patient selection for autotransplantation.

OBJECTIVE: To assess the efficiency of two vitrification protocols to cryopreserve human preantral follicles with the use of a xenografting model. DESIGN: Pilot study. SETTING: Gynecology research unit in a university hospital. PATIENT(S): Ovarian biopsies were obtained from seven women aged 30-41 years. INTERVENTION(S): Ovarian tissue fragments were subjected to one of three cryopreservation protocols (slow freezing, vitrification protocol 1, and vitrification protocol 2) and xenografted for 1 week to nude mice. MAIN OUTCOME MEASURE(S): The number of morphologically normal follicles after cryopreservation and grafting and fibrotic surface area were determined by histologic analysis. Apoptosis was assessed by the TUNEL method. Morphometric analysis of TUNEL-positive surface area also was performed. Follicle proliferation was evaluated by immunohistochemistry. RESULT(S): After xenografting, a difference was observed between the cryopreservation procedures applied. According to TUNEL analysis, both vitrification protocols showed better preservation of preantral follicles than the conventional freezing method. Moreover, histologic evaluation showed a significantly higher proportion of primordial follicles in vitrified (protocol 2)-warmed ovarian tissue than in frozen-thawed tissue. The proportion of growing follicles and fibrotic surface area was similar in all groups. CONCLUSION(S): Vitrification procedures appeared to preserve not only the morphology and survival of preantral follicles after 1 week of xenografting, but also their ability to resume folliculogenesis. In addition, vitrification protocol 2 had a positive impact on the quiescent state of primordial follicles after xenografting.

PURPOSE: To assess follicular growth after xenografting in order to understand how freezing and/or grafting may affect follicular development. METHODS: Human ovarian biopsies were used for fresh and frozen-thawed xenografting to SCID mice. After xenotransplantation, follicular morphology and proportion, oocyte and follicle diameter, and quantitative and qualitative parameters of antral follicles were analyzed. RESULTS: The proportion of growing follicles was significantly higher in grafted than non-grafted ovarian tissue. Follicular growth to the antral stage was observed and there was no significant difference in oocyte or follicle diameter in fresh or frozen-thawed grafts. Although no significant difference was observed in antral area or zona pellucida thickness, the theca layer in antral follicles from frozen-thawed grafted tissue was found to be significantly thinner than in fresh grafts. CONCLUSION: Antral follicles obtained after grafting of frozen-thawed human ovarian tissue showed a thinner theca cell layer compared to those from fresh grafts, which could affect follicular development and function. Further studies are nevertheless warranted to confirm the identity of theca cells and assess if they retain the ability to respond to luteinizing hormone and produce androgens.

OBJECTIVE: To evaluate the adverse effects of endometriomas on ovarian reserve. DESIGN: Analysis of prospectively collected biopsy samples. SETTING: Gynecology research unit in a university hospital. PATIENT(S): Women younger than age 35 years with endometriomas. INTERVENTION(S): Biopsy of normal cortex from ovaries affected by endometriomas (≤4 cm) and contralateral ovaries without cysts. MAIN OUTCOME MEASURE(S): Presence of cortex-specific stroma, observation of superficial endometriosis, follicular density, and presence of fibrosis. RESULT(S): Twenty samples of cortical tissue from ovaries with endometriomas and 11 from contralateral ovaries without cysts were analyzed. Follicular density was significantly lower in cortex from ovaries with endometriomas than in cortex from contralateral ovaries without cysts (mean ± SD = 6.3 ± 4.1/mm(3) vs 25.1 ± 15.0/mm(3)). Eleven (55%) cortical samples from ovaries with endometriomas showed fibrosis and concomitant loss of cortex-specific stroma, not observed in contralateral normal ovaries. Multivariate analysis revealed that the presence of endometrioma and fibrosis were significantly and independently associated with follicular density. CONCLUSION(S): Endometriotic cyst formation and associated structural tissue alterations in apparently normal ovarian cortex may be a cause of reduced ovarian reserve. Early diagnosis and intervention may be beneficial in women with endometriomas to protect their ovarian function.

To set up a protocol to isolate human preantral follicles with an enzyme produced in good manufacturing practice conditions for use in a clinical setting.For follicle isolation, ovarian biopsies were divided into two halves: one was treated with collagenase IA and the other with Liberase DH (Dispase High) Research Grade.Academic research unit.Twelve women undergoing laparoscopy for benign gynecological disease.Follicle isolation.Follicles were counted, their morphology was analyzed, and follicular viability was evaluated by live/dead assays before and after 7 days of in vitro culture. Their structural preservation was assessed after isolation by electron microscopy.A total of 1,030 follicles were obtained after isolation: 566 with Liberase DH and 464 with collagenase IA. The percentage of viable follicles (with10% of dead granulosa cells [GC]) was not found to be statistically different before or after culture in either group (Liberase DH: 95% and 81%, respectively; collagenase: 96% and 87%, respectively). A significant increase in follicle size was observed in both groups after culture. Liberase DH did not affect the ultrastructure of isolated follicles.Liberase DH allows isolation of a high number of preantral follicles, maintaining their viability, even after in vitro culture, and their ultrastructure. In addition, Liberase DH can be produced in good manufacturing practice conditions, allowing use of isolated preantral follicles for future clinical applications.

Objective To report a live birth after orthotopic transplantation of cryopreserved ovarian tissue. Design Case report. Setting Department of Gynecology, Cliniques Universitaires Saint-Luc, Brussels. Patient(s) Woman with metastatic cancer who had her ovarian tissue cryopreserved in 2001 before undergoing chemotherapy and hematopoietic stem cell transplantation, resulting in premature ovarian failure. Intervention(s) Orthotopic reimplantation of ovarian cortex performed 7 years after cryopreservation. Main Outcome Measure(s) Restoration of ovarian activity. Result(s) Restoration of ovarian activity was observed 3.5 months after reimplantantation, and ongoing pregnancy was diagnosed 9 months after grafting. The patient delivered a healthy baby weighing 2.830 kg. Conclusion(s) Our patient represents the thirteenth live birth to occur after orthotopic reimplantation of cryopreserved tissue, but the first in a woman treated for metastatic disease.

Because of the simplicity of vitrification, many authors have investigated it as an alternative to slow freezing for cryopreserving ovarian tissue. In the last decade, numerous studies have evaluated 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 appear 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 the utilization of different approaches to assess tissue functionality and oocyte quality is essential in order to validate the promising results already obtained with vitrification procedures. This review summarizes the principles of vitrification, discusses the advantages of vitrification protocols for ovarian tissue cryopreservation and describes different studies conducted on the vitrification of ovarian tissue in humans and animal species.

OBJECTIVE: To compare early follicular growth after fresh and frozen-thawed human ovarian tissue xenografting and to investigate whether expression of c-kit, kit ligand (KL), and growth differentiation factor-9 (GDF-9) is maintained after freezing and xenografting of human ovarian tissue. DESIGN: Prospective experimental study. SETTING: Gynecology research unit in a university hospital. ANIMAL(S): Ten nude (Swiss nu/nu) 6-week-old female mice. INTERVENTION(S): The ovarian biopsy samples were obtained from six women, aged 20 to 30 years. Fresh and frozen-thawed ovarian fragments intraperitoneally grafted into nude mice for 3 weeks. MAIN OUTCOME MEASURE(S): Histologic analysis and immunohistochemical evaluation of c-kit, KL, and GDF-9 expression before and after grafting. RESULT(S): The integrity and proportion of growing follicles increased similarly in both fresh (64%) and frozen-thawed (59%) xenografts compared with non-grafted tissue (fresh: 40%; frozen-thawed: 21%). Both C-kit and KL staining were detected in the oocytes and granulosa cells of preantral follicles, and GDF-9 expression was observed in the oocytes of preantral follicles in all groups. CONCLUSION(S): Freezing does not appear to have a major impact on early follicular growth after transplantation. This study shows, for the first time, expression of c-kit, KL, and GDF-9 in human preantral follicles after ovarian tissue cryopreservation and xenotransplantation.

Advances in the diagnosis and treatment of childhood, adolescent and adult cancer have greatly increased the life expectancy of premenopausal women with cancer. The ovaries are very sensitive to cytotoxic treatment, especially to alkylating agents. The only established method of fertility preservation is embryo cryopreservation according to the Ethics Committee of the American Society for Reproductive Medicine (2005), but this option requires the patient to be of pubertal age, have a partner or use donor sperm and be able to undergo a cycle of ovarian stimulation, which is not possible when the chemotherapy has to be initiated immediately or when stimulation is contraindicated, according to the type of cancer. For patients who need immediate chemotherapy, cryopreservation of ovarian tissue is the only possible alternative. This article reports the techniques and results of orthotopic transplantation of cryopreserved ovarian tissue. Among almost 30 cases reported in the literature, six live births have been achieved to date.

BACKGROUND Ovarian tissue cryopreservation is a promising technique to safeguard fertility in cancer patients. However, in some types of cancer, there is a risk of transmitting malignant cells present in the cryopreserved tissue. To avoid such a risk, pre-antral follicles could be isolated from ovarian tissue and grown in vitro. On the basis of this assumption, the aim of our study was to investigate in vitro survival and growth of pre-antral follicles after cryopreservation of ovarian tissue and follicular isolation, followed by encapsulation in alginate beads. METHODS Ovarian biopsies from four patients were frozen and thawed. Pre-antral follicles were then isolated and embedded in an alginate matrix before in vitro culture for 7 days. RESULTS Small pre-antral follicles (42.98 +/- 9.06 microm) from frozen-thawed tissue can survive and develop after enzymatic isolation and in vitro culture. A total of 159 follicles were incubated in a three-dimensional system (alginate hydrogel), and after 7 days, all of them showed an increase in size (final size 56.73 +/- 13.10 microm). The survival rate of the follicles was 90% (oocyte and all granulosa cells viable). CONCLUSION Our preliminary results indicate that alginate hydrogels may be a suitable system for in vitro culture of isolated human pre-antral follicles. However, more studies are required to establish whether follicular morphology and functionality can be maintained using this matrix.

BACKGROUND: Preservation of the male germ line in prepubertal boys undergoing gonadotoxic treatment is a crucial consideration in terms of their future quality of life. As these patients do not yet produce spermatozoa for freezing, only immature tissue is available for storage. We studied the survival, proliferation and differentiation capacity of spermatogonia after cryopreservation and long-term transplantation of immature testicular tissue pieces. METHODS: Single pieces of testicular tissue (2-8 mm(3)) from prepubertal boys (7-14 years) were cryopreserved, thawed and transplanted into the scrotum of mice for 6 months. Upon removal, histological, immunohistochemical and ultrastructural analyses and testicular sperm extraction (TESE) were used to evaluate the tissue. RESULTS: Histology showed 55 +/- 42% of tubules to be intact. MAGE-A4 immunostaining showed mean spermatogonial recovery to be 3.7 +/- 5.5%, with 35% of these cells expressing Ki67, evidencing proliferation in tissue from boys

Small human pre-antral follicles can be enzymatically isolated from the surrounding stroma, and are able to survive after 7 days of xenografting. The aim of the present study was to assess the developmental capacity of enzymatically isolated human follicles after long-term xenografting to severe combined immunodeficient (SCID) mice. Ovarian biopsies were obtained from three women 26-29 years of age. Human ovarian tissue was enzymatically dissociated using collagenase or a purified collagenase blend to obtain isolated follicles that were xenografted to SCID mice for 5 months. Recombinant FSH was given to the mice for the last 2 weeks. Five months after xenografting, follicular morphology was assessed by histology, and follicular proliferation by Ki-67 immunohistochemistry. Four grafts containing a total of 84 follicles were recovered. This follicular population was composed of 11 primordial follicles, 38 primary follicles, 31 secondary follicles and four antral follicles. Ki-67 was found to stain granulosa cells in antral follicles intensively. The results demonstrate, for the first time, that isolated human follicles are able to survive after long-term xenografting, and can develop into antral follicles after FSH stimulation.

During the last decade, new technologies in reproductive medicine have emerged to preserve the fertility of women whose gonadal function is threatened by premature menopause or gonadotoxic treatments. To offer an individualized approach to these patients, different experimental procedures are under investigation, including oocyte cryopreservation and cryopreservation and transplantation of ovarian tissue in the form of cortical fragments, whole ovary or isolated follicles. This review shows that transmission electron microscopy (TEM), combined with other in-vivo and in-vitro analysis techniques, is a valuable tool in the establishment of new experimental protocols to preserve female fertility. Ultrastructural studies allow in-depth evaluation of the oocyte's unique morpho-functional characteristics, which explain its low cryotolerance, and provide essential information on follicular, stromal and endothelial cell integrity, as well as cellular interactions crucial for normal folliculogenesis. In order to be able to offer appropriate and efficient options in every clinical situation, oocyte in-vitro maturation and ovarian tissue transplantation need to be optimized. Further development of new approaches, such as follicular isolation and whole ovary transplantation, should be encouraged. Fine ultrastructural details highlighted by TEM studies will be useful for the further optimization of these emerging technologies.

Endometriosis is characterized by pelvic inflammation that shows an increased number of activated peritoneal macrophages and their secreted products such as cytokines, growth factors, and angiogenic factors. Our results show that activation of nuclear factor-kappa B (NF-kappaB), a pro-inflammatory transcription factor, is statistically significantly increased in peritoneal macrophages from patients with endometriosis when compared with controls.

BACKGROUND: Fertility preservation has become an urgent clinical requisite for prepubertal male cancer patients undergoing gonadotoxic treatment. As these patients do not yet produce spermatozoa for freezing, only immature tissue is available for storage. We studied the survival and proliferative activity of spermatogonia and Sertoli cells after cryopreservation of cryptorchid testicular tissue pieces followed by xenografting for 21 days. METHODS AND RESULTS: Single pieces of tissue from cryptorchid testes (2-9 mm(3)) of young boys (2-12 years) were cryopreserved, thawed and transplanted into the scrotum of mice. Quantitative morphometric and immunohistochemical techniques were used to evaluate the integrity of the tissue, as well as the survival and proliferative capacity of spermatogonia and Sertoli cells before and after freezing/thawing/grafting. Three weeks after grafting, cryopreserved tissue was removed and analysed. Most of the tubules (88.3%) were intact and there was no fibrosis or sclerosis, 14.5% of the initial spermatogonial population remained, as identified by the MAGE A4 antibody, and 32% of these cells showed proliferative activity evidenced by Ki67, compared to 17.8% before cryopreservation and grafting. The number of Sertoli cells was unchanged and 5.1% were Ki67-positive, compared to none at all before freezing and grafting. CONCLUSIONS: Through our orthotopic xenografting model, we have demonstrated the survival and proliferative activity of spermatogonia and Sertoli cells in cryopreserved immature human cryptorchid tissue. Testicular tissue banking may thus prove to be a promising technique for the preservation of fertility in prepubertal boys undergoing oncological treatments. As the stem cell niche is maintained, the cryopreserved tissue can potentially be used for future autotransplantation. In addition, whole tissue freezing does not exclude alternative clinical uses, including isolated cell transplantation after dissociation, selection and enrichment. However, as this work was done on cryptorchid tissue, studies on normal immature testicular tissue, involving longer grafting periods, are needed to demonstrate a differentiation capacity before clinical implementation. Ethical and safety issues should also be addressed.

Red (active), black and white endometriotic lesions are characteristic of peritoneal endometriosis. The transcription factor nuclear factor-kappa B (NF-kappaB) activates proinflammatory, proliferative and antiapoptotic genes in many cell types. To determine whether NF-kappaB is activated in peritoneal endometriosis in women, and further ascertain the differential inflammatory status of endometriotic implants, NF-kappaB activation and intercellular adhesion molecule (ICAM)-1 expression were investigated in peritoneal endometriotic lesions according to their type. Furthermore, p65 and p50 subunits of active NF-kappaB dimers were evaluated in endometriotic lesions to gain some insight into NF-kappaB-implicated pathways. Thirty-six biopsies of peritoneal endometriotic lesions were analyzed. Constitutive NF-kappaB activation, involving p65- and p50-containing dimers, was demonstrated in peritoneal endometriotic lesions by electrophoretic mobility shift assays and supershift analyses, as well as NF-kappaB (p65) DNA-binding activity immunodetection assays. NF-kappaB activation and ICAM-1 expression (evaluated by immunoblotting) were significantly higher in red lesions than black lesions, whereas IkappaBalpha (NF-kappaB inhibitory protein) expression was constant, as shown by western blot analysis. This is the first study to demonstrate constitutive NF-kappaB activation in peritoneal endometriosis in women. NF-kappaB activation and ICAM-1 expression in red lesions confirm the more extensive inflammatory pattern of these lesions compared with black lesions. The involvement of p50/p65 dimers in NF-kappaB activation suggests implication of the classic NF-kappaB activation pathway, making it an attractive therapeutic target in endometriosis.

OBJECTIVE: To investigate the chorioallantoic membrane (CAM) model for the study of short-term transplantation of frozen human ovarian tissue. DESIGN: Prospective study. SETTING: Academic research unit. PATIENT(S): Ovarian tissue was obtained from three women. INTERVENTION(S): Frozen-thawed human cortical fragments were grafted onto traumatized CAM or beneath the CAM of 10-day-old chick embryos. Grafts were retrieved after 1, 2, 3, 4, and 5 days in ovo. MAIN OUTCOMES MEASURE(S): Viability was assessed by calcein-AM and ethidium homodimer I. Tissue integrity, ischemic injury, and neovascularization were evaluated by histology. Cell proliferation was analyzed by Ki-67 immunohistochemistry. RESULT(S): All the grafts showed adhesion when placed onto CAM, compared with only 30.4% beneath the CAM. Follicles were healthy, apart from a few degenerated follicles in necrotic and fibrotic areas. After 5 days, the majority of follicles were intermediate (32%) or primary (45.7%). Ki-67 immunohistochemistry revealed 12.5% proliferative follicles on day 2, reaching 20.7% on day 5. Fibrosis appeared on day 1; necrosis, follicular degeneration and follicular proliferation on day 2; and neovascularization and stromal cell proliferation on day 3. CONCLUSION(S): The present study showed that the CAM model provides a new approach to study human ovarian tissue transplantation in its first ischemic stages, yielding information on the timing of tissue changes before the establishment of neovascularization.

Background: In vitro studies suggest that the transcription factor nuclear factor-kappa B (NF-ĸB) is implicated in the transduction of proinflammatory signals in endometriosis. The aim of this study was to investigate the involvement of NF-ĸB and the processes regulated by NF-ĸB in the initial development of endometriotic lesionsin vivo.Methods: Endometriosis was induced in nude mice by intraperitoneal injection of fluorescent-labeled menstrual endometrium. Two NF-ĸB inhibitors (BAY 11-7085 and SN-50) were injected intraperitoneally on days 0, 2 and 4 after endometriosis induction, and endometriotic lesions were recovered on day 5. Number, mass, fluorimetry and surface (morphometry) of endometriotic lesions were quantified. NF-ĸB activation, intercellular adhesion molecule (ICAM)-1 expression, cell proliferation and apoptosis were evaluated by immunohistochemical analyses and the TUNEL method. Results: Both NF-ĸB inhibitors induced a significant reduction in lesion development compared to control mice. NF-ĸB activation and ICAM-1 expression of endometriotic lesions were significantly reduced in treated mice, and cell proliferation was significantly reduced in BAY 11-7085-treated mice. Both inhibitors produced a significant increase in apoptosis of endometriotic lesions, as assessed by active caspase-3 immunostaining and the TUNEL method. Conclusion: This study demonstrates, for the first time, that the NF-ĸB pathway is implicated in the development of endometriotic lesions in vivo and that NF-ĸB inhibition reduces ICAM-1 expression and cell proliferation, but increases apoptosis of endometriotic lesions, diminishing the initial development of endometriosis in an animal model.

OBJECTIVE: To analyze the ultrastructure of human ovarian follicles after cryopreservation and short-term xenografting. DESIGN: Prospective experimental study. SETTING: Academic gynecology and anatomy research units. PATIENT(S): Ovarian cortical biopsy specimens were obtained from 13 patients. INTERVENTION(S): Each ovarian biopsy specimen was dissected into pieces of 1 mm(3) and divided into three groups: [1] fresh tissue, [2] frozen-thawed tissue, and [3] frozen-thawed tissue xenografted onto the peritoneum of nude mice for 3 weeks. MAIN OUTCOME MEASURE(S): Follicular ultrastructure was assessed by light and transmission electron microscopy in [1] fresh, [2] frozen, and [3] frozen-grafted tissue. RESULT(S): Thirty-five ovarian follicles were analyzed by light and transmission electron microscopy. Twenty-five primordial and primary ovarian follicles were found. Most of them exhibited ultrastructurally well preserved features (fresh [N = 8/10], frozen [N = 7/10], and frozen-grafted [N = 4/5] tissue). Ten secondary follicles were present in xenografts. By transmission electron microscopy, all the healthy-looking secondary follicles (70%) were shown to contain intact oocytes, with features typical of earlier developmental stages, surrounded by several layers of follicular cells. CONCLUSION(S): The present study demonstrates, for the first time, that cryopreservation and xenotransplantation do not appear to greatly affect human primordial/primary follicle ultrastructure. Interestingly, in frozen-thawed xenografts, secondary human ovarian follicles presented a well preserved ultrastructure, but asynchrony between oocyte and granulosa cell development was detected. The possible causes for this asynchrony are discussed.