Outcomes of immature oocytes collected from ovarian tissue for cryopreservation in adult and prepubertal patients.

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-Müllerian 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.

Endometriosis is associated with aberrant metabolite profiles in plasma.

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.

In Search of Better Spermatogonial Preservation by Supplementation of Cryopreserved Human Immature Testicular Tissue Xenografts with N-acetylcysteine and Testosterone.

Controlled slow-freezing is the procedure currently applied for immature testicular tissue (ITT) cryobanking in clinical practice. Vitrification has been proposed as a promising alternative, with a view to better preserve the spermatogonial stem cells for future fertility restoration by autografting in young boys suffering from cancer. It appears that besides the potential influence of the cryopreservation technique used, the transplantation procedure itself has a significant impact on spermatogonial loss observed in ITT xenografts. Eighteen ITT pieces issued from 6 patients aged 2-15 years were used. Fragments of fresh tissue (serving as ungrafted controls), frozen-thawed tissue, frozen-thawed tissue supplemented with N-acetylcysteine (NAC), and frozen-thawed tissue supplemented with testosterone xenografted to nude mice for 5 days were compared. Upon graft removal, histological and immunohistochemical analyses were performed to evaluate spermatogonia, intratubular proliferation, and intrinsic and extrinsic apoptosis. A significant decrease in the integrity of intact seminiferous tubules was found in all three grafted groups. Spermatogonia were observed by immunohistochemistry in all grafted groups, with recovery rates of 67, 63, and 53%, respectively, for slow-frozen tissue, slow-frozen tissue supplemented with NAC, and slow-frozen tissue supplemented with testosterone. Apoptosis evidenced by active caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was similar in all grafts. The study is limited by the low availability of ITT samples of human origin, and no clear impact of graft supplementation was found. The mouse xenotransplantation model needs to be refined to investigate human spermatogenesis in human ITT grafts.

Differential expression of steroidogenic enzymes according to endometriosis type.

OBJECTIVE: To evaluate, in peritoneal, ovarian, and rectovaginal endometriotic lesions, expression of steroidogenic enzymes involved in the activation and inactivation of estrogens: 17ß-hydroxysteroid dehydrogenase type 1 (HSD17B1) and 2 (HSD17B2), estrone sulfotransferase (EST), and steroid sulfatase (STS). SETTING: Academic gynecology research unit. DESIGN: Retrospective study. PATIENT(S): 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. INTERVENTION(S): Q-PCR and immunohistochemistry. MAIN OUTCOME MEASURE(S): Evaluation of mRNA and protein expression. RESULT(S): 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. CONCLUSION(S): In endometriosis development, local activation of estrogens appears to be important. STS and HSD17B1 inhibitors may therefore prove useful to treat the disease.

Nerve fiber density in deep nodular endometriotic lesions induced in a baboon experimental model.

OBJECTIVE: To study the occurrence of nerve fibers in deep nodular endometriotic lesions after nodules were induced in baboons and nerve fiber densities measured 6 months after the grafting procedure. DESIGN: Experimental animal study. SETTING: Academic gynecology research unit. ANIMALS: Ten baboons (Papio anubis). INTERVENTION(S): Recovery of induced endometriotic nodules and eutopic endometrium. MAIN OUTCOME MEASURE(S): Protein gene product (PGP) 9.5 and nerve growth factor (NGF) immunohistochemistries were performed to evaluate nerve fiber density and NGF expression in induced endometriotic lesions and eutopic endometrium. RESULT(S): Eutopic (basalis) endometrium, myometrium, and invasive and noninvasive nodular lesions were analyzed separately. The highest nerve fiber densities were observed in normal myometrium and in the basal layer of eutopic endometrium. No significant differences were observed between the two lesion types. However, the NGF staining intensity score was found to be higher in glands of deep invasive lesions than in glands of eutopic baboon endometrium. CONCLUSION(S): This is the first study to show the presence of nerve fibers in eutopic baboon endometrium and induced deep endometriotic nodules. Long-term studies are now warranted to determine if nerves still grow in invasive and noninvasive lesions >6 months after grafting, and to evaluate the role of the lesion environment.

Successful vitrification and autografting of baboon (Papio anubis) ovarian tissue.

STUDY QUESTION: Can a vitrification protocol using an ethylene glycol/dimethyl sulphoxide-based solution and a cryopin successfully cryopreserve baboon ovarian tissue? SUMMARY ANSWER: Our results show that baboon ovarian tissue can be successfully cryopreserved with our vitrification protocol. WHAT IS KNOWN ALREADY: Non-human primates have already been used as an animal model to test vitrification protocols for human ovarian tissue cryopreservation. STUDY DESIGN, SIZE, DURATION: Ovarian biopsies from five adult baboons were vitrified, warmed and autografted for 5 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS: 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. LIMITATIONS, REASONS FOR CAUTION: Despite our promising findings, before applying this technique in a clinical setting, we need to validate it by achieving pregnancies. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTEREST(S): 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.

Should we isolate human preantral follicles before or after cryopreservation of ovarian tissue?

OBJECTIVE: To evaluate the survival and growth potential of human preantral follicles isolated before and after cryopreservation. DESIGN: Pilot study. SETTING: Gynecology research unit in a university hospital. PATIENT(S): Six women aged 27 to 32 years. INTERVENTION(S): Six ovarian biopsy samples were cut into two equal parts, half subjected to slow-freezing followed by follicle isolation (cryo-iso group) and alginate-matrigel embedding, and half immediately processed for follicle isolation and alginate-matrigel embedding followed by slow-freezing (iso-cryo group) or used as fresh controls (fresh group). MAIN OUTCOME MEASURE(S): Follicle number, viability, diameter, and morphology. RESULT(S): After 1,134 preantral follicles had been isolated from fresh biopsy samples and 1,132 from frozen specimens, the three groups were compared before and after 7 days of in vitro culture (IVC) in alginate-matrigel beads. No statistically significant differences in viability were found between the three groups before or after IVC, but follicle diameter increased in all three groups after IVC. Morphology analysis revealed well-preserved follicles in both the iso-cryo and cryo-iso groups after IVC. CONCLUSION(S): Human preantral follicles can be successfully cryopreserved before or after isolation without impairing their ability to survive and grow in vitro. This could lead to development of new protocols for follicle cryopreservation, IVC, and grafting in clinical and research settings for fertility preservation.

A review of 15 years of ovarian tissue bank activities.

PURPOSE: To review 15 years of activities in ovarian tissue cryobanking from medical database files, including patient indications, histological evaluation and clinical characteristics. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Vitrification preserves proliferation capacity in human spermatogonia.

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 (3ß-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 Télévie N° 7. 4.572.09.F). The authors declare that there is no conflict of interest.

Induction of endometriotic nodules in an experimental baboon model mimicking human deep nodular lesions.

OBJECTIVE: To establish an experimental model for the study of deep nodular endometriosis. DESIGN: Induction of nodular endometriosis in baboons by grafting different uterine specimens to the peritoneal cavity. SETTING: Research and university facilities. ANIMAL(S): Ten baboons, to develop a model of induced deep nodular endometriosis. INTERVENTION(S): Biopsies of endometrium, and endometrium plus the junctional zone (JZ), full uterine thickness, and myometrium grafted to the peritoneum. MAIN OUTCOME MEASURE(S): Macroscopic descriptions recorded for observed induced lesions; staining with hematoxylin and eosin for histological evaluation and specific antibodies (CK22, CD10) for immunohistochemical studies; and analysis of surface area and volume of lesions, glandular density, and invasion of surrounding organs. RESULT(S): The incidence of induced nodular endometriosis was 100%, but the extent depended on the tissue grafted. Lesions induced after grafting specimens containing the JZ were statistically significantly larger than those not containing the JZ. Surrounding organ invasion was reported in more than 40% of lesions after grafting specimens containing the JZ. CONCLUSION(S): The first experimental model of nodular endometriosis allows investigation of deeper nodular lesions as well as invasion phenomena associated with nodular lesions.

Vitrification and xenografting of human ovarian tissue.

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.

Transplantation of an alginate-matrigel matrix containing isolated ovarian cells: first step in developing a biodegradable scaffold to transplant isolated preantral follicles and ovarian cells.

For women diagnosed with leukemia, transplantation of cryopreserved ovarian tissue after disease remission is not advisable. Therefore, to restore fertility in these patients, we aim to develop a biodegradable artificial ovary that offers an environment where isolated follicles and ovarian cells (OCs) can survive and grow. Four NMRI mice were ovariectomized and their ovaries used to isolate OCs. Groups of 50,000 OCs were embedded in an alginate-matrigel matrix for further fixation (fresh controls), one week of in vitro culture (IVC) or heterotopic autografting. OC proliferation (Ki67), apoptosis (TUNEL), scaffold degradation, vessel formation (CD34) and inflammation (CD45) were analyzed. Ki67-positive OCs were found in 2.3%, 9.0% and 15.5% cells of cases in fresh, IVC and grafted beads respectively, while cells were TUNEL-positive in 0%, 1.5% and 6.9% of cases. After IVC or grafting, the beads degraded, losing their original round aspect, and infiltrating blood capillaries could be observed in the grafted beads. CD34-positive cells and 22% CD45-positive cells were found around and inside the matrix. In conclusion, our results demonstrate that an alginate-based matrix is a promising proposition to graft isolated OCs. After transplantation, this matrix was able to degrade, allowed vascularization and elicited a low inflammatory response.

Effect of cryopreservation and transplantation on the expression of kit ligand and anti-Mullerian hormone in human ovarian tissue.

BACKGROUND: Although 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-Müllerian hormone (AMH), two key factors activating and inhibiting follicle growth, were assessed after long-term grafting in severe combined immunodeficient (SCID) mice. METHODS: Ovarian 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). RESULTS: Lower 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. CONCLUSIONS: Grafting 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.

Peritoneal endometriosis is an inflammatory disease.

Peritoneal endometriosis is a chronic inflammatory disease characterized by increased numbers of peritoneal macrophages and their secreted products. Inflammation plays a major role in pain and infertility associated with endometriosis, but is also extensively involved in the molecular processes that lead to peritoneal lesion development. Peritoneal oxidative stress is currently thought to be a major constituent of the endometriosis-associated inflammatory response. Excessive production of reactive oxygen species, secondary to peritoneal influx of pro-oxidants such as heme and iron during retrograde menstruation, may induce cellular damage and increased proinflammatory gene expression through nuclear factor-kappa B activation. In particular, prostaglandin biosynthetic enzyme expression is regulated by this transcriptional factor, and increased peritoneal prostaglandin concentrations have been demonstrated in endometriosis. In the light of available data collected from patient biopsies, as well as in vitro and in vivo studies, the respective involvement and potential molecular interactions of iron, nuclear factor-kappa B and prostaglandins in the pathogenesis of endometriosis are explored and discussed. The key role of peritoneal macrophages is emphasized and potential therapeutic targets are examined.

Impact of freezing and thawing of human ovarian tissue on follicular growth after long-term xenotransplantation.

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.

Endometriomas as a possible cause of reduced ovarian reserve in women with endometriosis.

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.

In vivo biocompatibility of three potential intraperitoneal implants.

The intraperitoneal biocompatibility of PDMS, polyHEMA and pEVA was investigated in rats, rabbits and rhesus monkeys. No inflammation was evidenced by hematological analyses and measurement of inflammatory markers throughout the experiment and by post-mortem examination of the pelvic cavity. After 3 or 6 months, histological analysis revealed fibrous tissue encapsulating PDMS and PEVA implants in all species and polyHEMA implants in rabbits and monkeys. Calcium deposits were observed inside polyHEMA implants. The intraperitoneal biocompatibility of all 3 polymers makes them suitable for the design of drug delivery systems, which may be of great interest for pathologies confined to the pelvic cavity.

Enzymatic isolation of human primordial and primary ovarian follicles with Liberase DH: protocol for application in a clinical setting.

OBJECTIVE: 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. DESIGN: 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. SETTING: Academic research unit. PATIENT(S): Twelve women undergoing laparoscopy for benign gynecological disease. INTERVENTION(S): Follicle isolation. MAIN OUTCOME MEASURE(S): 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. RESULT(S): A total of 1,030 follicles were obtained after isolation: 566 with Liberase DH and 464 with collagenase IA. The percentage of viable follicles (with <10% 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. CONCLUSION(S): 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.

Is the baboon model appropriate for endometriosis studies?

OBJECTIVE: To determinethe prevalence of spontaneous endometriosis andthe incidence of induced endometriosis after endocervical canal resection in baboons. DESIGN: Induction and follow-up of endometriosis in baboons, which is one of the primate species that develop spontaneous endometriosis. Forty-one baboons were checked for the presence of spontaneous endometriosis. We then attempted to induce endometriosis in 30 of them by endocervical canal resection. SETTING: Institute of Primate Research, Nairobi, Kenya, and Catholic University of Louvain, Brussels, Belgium. ANIMAL(S): Forty-one baboons were checked for spontaneous endometriosis and 30 of them were used to develop a model of induced endometriosis. INTERVENTION(S): A total of 41 baboons underwent diagnostic laparoscopy for 10 months. In a first step, 30 of this number subsequently underwent endocervical canal resection. In a second step, 20 of the 30 underwent uterine horn resection. MAIN OUTCOME MEASURE(S): Follow-up by laparoscopy. RESULT(S): Two of the 41 baboons were diagnosed with spontaneous endometriosis (4.8%). Twelve months after the surgical procedure to induce endometriosis, 8 of 29 animals presented with endometriotic lesions diagnosed by using laparoscopy and confirmed by histologic examination. The incidence of induced endometriosis in our model was thus 27.6%. In 2 baboons, endometriosis disappeared over time, resulting in a final rate of 20.7% (6/29). CONCLUSION(S): The rate of spontaneous endometriosis is very low (4.8%). Endometriosis can be induced (with a rate of just 27.6%) by endocervical canal resection to stimulate retrograde menstruation.

Insights into iron and nuclear factor-kappa B (NF-kappaB) involvement in chronic inflammatory processes in peritoneal endometriosis.

Endometriosis is a chronic pelvic inflammatory process. Local inflammation is known to play a role in pain and infertility associated with the disease, and may be extensively involved in molecular and cellular processes leading to endometriosis development. In this review, we focus on two inflammatory mediators clearly implicated in the pathogenesis of endometriosis, iron and NF-kappaB, and their potential association. Iron is essential for all living organisms, but excess iron results in toxicity and is linked to pathological disorders. In endometriosis patients, iron overload has been demonstrated in the different compartments of the peritoneal cavity (peritoneal fluid, endometriotic lesions, peritoneum and macrophages). This iron overload affects numerous mechanisms involved in endometriosis development. Moreover, iron can generate free radical species able to react with a wide range of cellular constituents, inducing cellular damage. Overproduction of reactive oxygen species also impairs cellular function by altering gene expression via regulation of redox-sensitive transcription factors such as NF-kappaB, which is clearly implicated in endometriosis. Indeed, NF-kappaB is activated in endometriotic lesions and peritoneal macrophages of endometriosis patients, which stimulates synthesis of proinflammatory cytokines, generating a positive feedback loop in the NF-kappaB pathway. NF-kappaB-mediated gene transcription promotes a variety of processes, including endometriotic lesion establishment, maintenance and development. In conclusion, iron and NF-kappaB appear to be linked and both are clearly involved in endometriosis development, making these pathways an attractive target for future treatment and prevention of this disease.