Belen Martinez-Madrid

Livebirth after orthotopic transplantation of cryopreserved ovarian tissue

BACKGROUND The lifesaving treatment endured by cancer patients leads, in many women, to early menopause and subsequent infertility. In clinical situations for which chemotherapy needs to be started, ovarian tissue cryopreservation looks to be a promising option to restore fertility. In 1997, biopsy samples of ovarian cortex were taken from a woman with stage IV Hodgkins lymphoma and cryopreserved before chemotherapy was initiated. After her cancer treatment, the patient had premature ovarian failure. METHODS In 2003, after freeze-thawing, orthotopic autotransplantation of ovarian cortical tissue was done by laparoscopy. FINDINGS 5 months after reimplantation, basal body temperature, menstrual cycles, vaginal ultrasonography, and hormone concentrations indicated recovery of regular ovulatory cycles. Laparoscopy at 5 months confirmed the ultrasonographic data and showed the presence of a follicle at the site of reimplantation, clearly situated outside the ovaries, both of which appeared atrophic. From 5 to 9 months, the patient had menstrual bleeding and development of a follicle or corpus luteum with every cycle. 11 months after reimplantation, human chorionic gonadotrophin concentrations and vaginal echography confirmed a viable intrauterine pregnancy, which has resulted in a livebirth. INTERPRETATION We have described a livebirth after orthotopic autotransplantation of cryopreserved ovarian tissue. Our findings suggest that cryopreservation of ovarian tissue should be offered to all young women diagnosed with cancer.

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.

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.

The role of cryopreservation for women prior to treatment of malignancy.

Purpose of review The purpose of this review is to investigate recent advances in xenografting, as well as in orthotopic and heterotopic autotransplantation of human cryopreserved ovarian tissue. Recent findings The first livebirth after orthotopic transplantation of cryopreserved ovarian tissue was reported recently. We discuss this case and other cases of reimplantation of cryopreserved ovarian tissue, bearing in mind that many questions remain. Summary Finally, we report the latest developments in research on the transplantation of an intact ovary and the reimplantation of isolated follicles.

Chick embryo chorioallantoic membrane (CAM) model: a useful tool to study short-term transplantation of cryopreserved human ovarian tissue

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.

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.

Quantification and viability assessment of isolated bovine primordial and primary ovarian follicles retrieved through a standardized biopsy pick-up procedure

The feasibility of repeated collection and enzymatic isolation of large numbers of viable primordial and primary follicles from living donor cows were tested. Ovarian cortical biopsies were collected transvaginally by the Biopsy Pick-Up (BPU) device, a modification of an Ovum Pick-Up instrument. Follicles were enzymatically isolated from the retrieved cortical tissue samples, and follicle viability was determined by a live/dead fluorescent assay. Six cows were subjected to BPU once per week during 4 consecutive weeks, and in each BPU session 4 cortical tissue samples were collected per ovary. Over the 4-week trial period, a total of 1443 primordial and primary follicles were collected, 1358 (94%) of which were primordial and 85 (6%) were primary follicles. In each BPU session, an average 60.1 +/- 10.7 (mean +/- SEM) primordial and primary follicles were isolated per cow. The number of follicles varied considerably throughout the trial period and between cows. Statistical analysis of the data, however, did not support the presence of any distinct trends in the follicle yields over time or between cows. A total of 111 enzymatically isolated follicles were analyzed for viability with fluorescent probes. The vast majority of isolated follicles (92.8%) were totally viable. We conclude that the standardized BPU procedure generates sufficiently large numbers of vital primordial and primary follicles, thus validating BPU as a new tool for research into early bovine follicular development.

Human endometrial epithelial cells (EEC) constitutively express more intercellular adhesion molecule (ICAM)-1 than endometrial stromal cells (ESC) in culture.

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).

Viability of enzymatically isolated bovine primordial and primary ovarian follicles collected by the Biopsy Pick-Up technique

Our institution recently developed a new tool for transvaginal, ultrasound-guided collection of ovarian biopsies from living donor cows (Aerts et al. 2005 Theriogenology 64, 947–957). The biopsy pickup (BPU) device consists of a modified needle guidance system, which is equipped with a trocar needle and carries a 60-cm-long true-cut biopsy needle. In a previous experiment, the presence of primordial and preantral follicles in BPU-derived biopsies was demonstrated. As follicular integrity is a prerequisite for further culture or research, the aim of the present study was to assess the viability of enzymatically isolated primordial and primary ovarian follicles collected by the BPU instrument. Four cows were subjected to a one-time BPU procedure. Four cortical biopsies were collected per ovary from each animal. Follicle viability was determined by the dual fluorescent probe technique described by Cortvrindt and Smitz (2001 Fertil. Steril. 75, 588–593). In live cells, calcein-AM is converted into calcein by intracellular esterase enzymes, producing an intense green fluorescence. In dead cells, ethidium homodimer-I is able to penetrate the damaged plasma membrane and, upon binding to nucleic acids, undergo a 40-fold enrichment of fluorescence, thereby producing an intense red signal. Upon retrieval, the biopsies were immersed in HEPES-buffered minimum essential medium (GIBCO, Grand Island, NY, USA) at 4°C. In the laboratory, the tissue samples were transferred to 50-mL conical tubes containing 20 mL PBS supplemented with 1 mg mL-1 collagenase type IA (Sigma-Aldrich NV/SA, Bornem, Belgium) and were incubated in a water bath at 37°C for 60 min. The digestion was terminated by adding an equal volume of cold PBS. The resulting suspension was centrifuged at 300g for 10 min at 8°C. The pellet was resuspended in PBS and transferred to a Petri dish. Each Petri dish was examined under a phase-contrast inverted microscope (Olympus CHX41) equipped with an eyepiece micrometer, and the primordial and primary follicles were collected with a glass micropipette. Isolated follicles were mounted on a slide in 50-µL droplets of PBS containing 2 µmol L-1 calceine-AM and 5 µmol L-1 ethidium homodimer-I (Molecular Probes, Leiden, The Netherlands), and incubated in the dark for 45 min at 37°C. After incubation, the follicles were examined under a fluorescence microscope (Olympus BX61). A total of 95 enzymatically isolated follicles were analyzed for viability with fluorescent probes; 89 (93.7%) of these were viable. Viable follicles were retrieved from all animals. This experiment confirms that viable primordial and primary follicles can be retrieved from living donor cows by the BPU procedure, which makes the technique suited for further culture or research.