Alessandra Camboni

Short-term transplantation of isolated human ovarian follicles and cortical tissue into nude mice

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 (P<0.001). Moreover, the histological aspect of isolated follicle grafts was similar to that of grafted fragments: follicles were surrounded by vimentin-positive stroma-like tissue of human origin, as confirmed by fluorescent in 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.

IVF outcome in patients with orthotopically transplanted ovarian tissue

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.

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.

Development of antral follicles after xenografting of isolated small human preantral follicles.

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.

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.

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

PurposeTo assess follicular growth after xenografting in order to understand how freezing and/or grafting may affect follicular development.MethodsHuman 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.ResultsThe 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.ConclusionAntral 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.

Preservation of fertility in young cancer patients: contribution of transmission electron microscopy

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 oocytes 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.

Alginate beads as a tool to handle, cryopreserve and culture isolated human primordial/primary follicles.

BACKGROUND One major concern of grafting cryopreserved ovarian tissue to restore fertility in cancer patients is the possibility of reintroducing tumor cells. Cryopreservation of isolated primordial/primary follicles (PFs) may circumvent this problem. The aim of our work was to compare dimethyl sulfoxide (ME2SO) and ethylene glycol (EG) as cryoprotectants (CPAs) for slow-freezing of isolated human PFs in alginate. METHODS Ovarian biopsies from four women were processed for follicle isolation. PFs were embedded in alginate (5-15 per group). Follicles were frozen-thawed using 1.4M ME2SO or 1.5M EG as CPAs. Fresh and cryopreserved isolated follicles were in vitro cultured (IVC) for 7 days. At different time periods (after isolation, cryopreservation and IVC), follicles were evaluated with live/dead assay (using fluorescent probes) and diameter measurement. Follicle viability was calculated according to the percentage of dead follicular cells and the presence of a live/dead oocyte. RESULTS A total of 841 PFs were isolated, embedded in alginate and cryopreserved with ME2SO (n=424) or EG (n=259), or used as controls (n=158). After 7 days of IVC, a significant increase in follicle size was observed in the fresh and ME2SO groups, but not in the EG group. The percentage of totally viable PFs was not significantly different before or after seven days of culture in fresh (100% and 82%) or ME2SO (93.2% and 85.1%) tissue. The EG group showed significantly lower viability before (63.9%) and after IVC (66.2%) than the fresh and ME2SO groups. CONCLUSIONS Our results show that 1.4M ME2SO yields better preservation of isolated PF viability after thawing and 7 days of IVC than 1.5M EG. Alginate constitutes an easy, safe hydrogel matrix to handle and cryopreserve isolated human follicles using ME2SO as a CPA.

A large polyp in the rectum: not always an epithelial lesion

Dear Editor:Solitaryextramedullaryplasmocytoma(SEP)isanexceeding-ly rare entity accounting for less than 5 % of all plasma celldyscrasias,suchasmonoclonalgammopathyofundeterminedsignificance (MGUS), multiple myeloma (MM), solitaryplasmocytoma of the bone, and monoclonal immunoglobulindeposition diseases. SEP is usually found in the upper respi-ratory tract and secondary in the gastrointestinal tract, withonly 6.2 % arising in the large bowel [1]. Only fifteen case ofano-rectal SEP were reported in the English literature.The diagnosis of SEP requires demonstration of a mono-clonal plasma cell infiltration in organs other than the bonemarrow without evidence of systemic involvement [2].SEP patients mainly manifest with local masses and rele-vant symptoms, which are nonspecific and depend on the siteandspreadofthetumor.InthelowGItract,obstruction,rectalbleeding, and abdominal pain were more commonlydescribed.Amongplasmacellneoplasia,SEPshowsthebestprogno-sis with 10-year overall survival rate of 70 %. Although atransformation into MM has been described in a small per-centage of case (∼15%), patients with SEP that progressedtoMM had a 100 % 5-year survival rate [3].We reported a case of rectal SEP in a 60-years-old manpresented with a 1 month of history of painless hematocheziaand diarrhea. Past medical and surgical history of ischemicheart disease and femur fracture was reported.At physical examination, no palpable abdominal or rectalmass were detected.Colonoscopy revealed one small polyp in the cecum andthree polyps in the rectum, two small and one of large size.Endoscopic mucosal resection (EMR) was performed for thelargest.At macroscopic examination, the large rectal polyp, of 4×3×2.5 cm of size, showed a smooth surface superficiallyulcerated.Thesurgical margin wasinkedinblack.Atsection,thespecimenshowedafleshyandmulticysticappearanceandwas involved in toto. All sections were processed for histo-logical analysis.Thethreesmallsizepolypsweretubuloadenomawithlowgrade dysplasia.Histological analysis of the largest polyp showed massiveinfiltration by medium-sized cells, characterized by abundantpale to eosinophilic cytoplasm and eccentric nuclei with acartwheel arrangement of the chromatin. Neither mitosis norapoptotic figures were found. No residual rectal glands werefound. Congo red staining showed no amyloid deposits.At immunohistochemistry, tumor cells revealed diffusereactivity for CD138 and for immunoglobulin kappa lightchains. Immunostaining for lambda light chains, CD20,CD45 were negative on tumoral cells. Ki67 showed a lowproliferation rate. Immunostaining for CD3 revealed someaccompanying Tcells CD3+.A diagnosis of rectal plasmocytoma was retained.As rectal plasmocytomas are extremely rare, reactiveplasmacytosis, plasma cell granuloma, and lymphoma withplasma cell differentiation (mucosa-associated lymphoid tis-sue (MALT), lymphoblastic, and immunoblastic) have to beexcluded in order to avoid misdiagnosis.

Primary central nervous system lymphoma of T-cell origin: an unusual cause of spinal cord disease

Less than 1% of lymphomas are primary central nervous system lymphomas (PCNSL) [1]. Most of them are B-cell lineage and involve the supratentorial space, whereas T-cell origin and involvement of spinal cord are extremely rare [1]. Despite its rarity, primary spinal cord lymphoma (PSCL) of T-cell origin requires a meticulous diagnosis, because delayed treatment can shorten survival. However, there are no typical clinical features or radiological findings and it is always necessary to perform biopsy to obtain accurate pathological diagnosis so as to guide therapeutical decisions and avoid severe neurological sequelae. Case report