Cortical Strips Research Articles

Live birth after ovarian tissue autograft in a patient with sickle cell disease treated by allogeneic bone marrow transplantation

To report the first case of restoration of ovarian activity and live birth after cryopreserved ovarian tissue autograft in a patient without cancer treated by allogeneic bone marrow transplantation. Case report. University hospital. One woman with homozygous sickle cell anemia. An orthotopic autotransplantation of ovarian cortical strips was performed after freeze-thawing. Cryopreservation of ovarian tissue, bone marrow transplantation, ovarian autograft, and restoration of ovarian function. In autumn 2005, biopsy samples of ovarian tissue were cryopreserved before chemotherapy followed by bone marrow transplantation. In spring 2008, because the patient had been menopausal for 2.5 years as a result of the conditioning therapy, an orthotopic autotransplantation of thawed ovarian cortex was performed. The patient conceived spontaneously in a natural cycle in autumn 2008, and delivered a healthy female child in June 2009. Cryopreservation of ovarian tissue with subsequent autotransplantation is an emerging procedure for preserving the fertility of young patients with a high risk of premature ovarian failure (POF) resulting from gonadotoxic treatment. This case opens up new perspectives in cases of nonmalignant diseases.

Successful Pregnancy After Allogeneic Bone Marrow Transplantation Followed by Ovarian Tissue Autograft in a Sickle Cell Patient.

Abstract 4482One major drawback of myeloablative allogeneic stem cell transplantation is the near 100% risk of profound fertility impairment leading to early menopause and sterility in most female patients. This toxic effect is especially critical in patients without malignant disease, i.e hemoglobinopathies or other inherited diseases. Cryopreservation of ovarian tissue with subsequent autotransplantation is an emerging procedure to preserve the fertility of young patients with a high risk of premature ovarian failure resulting from radiotherapy or chemotherapy for cancer.We report the restoration of ovarian activity and pregnancy with live birth after an orthotopic transplantation of frozen/thawed ovarian tissue in a 23 year old female patient given a transplant for sickle cell disease. In spring 2005, the patient of S/S hemoglobin genotype experienced a central nervous system stroke with unilateral central retinal artery occlusion. Monthly exchange transfusions were started from then and a pretransplant procedure was undertaken with a 10/10 matched sibling male donor who was heterozygous for HbS. In autumn 2005, biopsy samples of ovarian tissue were cryopreserved prior to conditioning. The patient received a conditioning regimen with IV busulfan (12.8 mg/kg total dose), cyclophosphamide (200 mg/kg total dose) and antilymphocyte globulin (15 mg/kg total dose) followed by allogeneic bone marrow transplantation (BMT). A stable 100% donor chimerism was obtained from D60.The patient developed grade II acute GVHD treated by steroids, then followed by limited chronic GVHD treated by a combination of mycophenolate-mofetil and ciclosporin. The treatment was tapered and stopped 7 months after BMT. After BMT, the patient presented clinical menopause with amenorrhoea and hot flushes due to premature ovarian failure. FSH and LH concentrations rose to menopausal levels. Hormone replacement therapy with oestro-progestogens was started 6 months after BMT and was maintained until ovarian function restoration after ovarian transplantation. In spring 2008, as the patient had been menopausal for 2.5 years as a result of the conditioning chemotherapy, an orthotopic autotransplantation of ovarian cortex was performed. The cortical fragments were grafted according to the following procedure: a first laparoscopy was performed to create a peritoneal windows between the right iliac vessels and an ovarian window in the remaining left ovary with the aim of further inducing angiogenesis. One thawed strip of ovarian cortex was cut in fragments, that were fixed in the ovarian incision and deposited in the peritoneal window. Three days later, a second laparoscopy was performed and three thawed cortical strips were fixed into the left ovary and one into the peritoneal window. Ovarian function recovery was evaluated by hormonal levels and follicular development was observed by ultrasound. The patient conceived spontaneously in a natural cycle in autumn 2008, and delivered a healthy female child in June 2009.This first case report of pregnancy and delivery after ovarian autograft in a patient treated by allogeneic BMT suggests that cryopreservation of ovarian tissue should be offered prior to SCT not only to women of childbearing age but also to prepubertal patients, as primordial follicles are already present in the post natal ovaries. This technique still deserves further investigations in leukemias but appears safe in non malignant diseases.Time line of treatment [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Potential indications for ovarian autotransplantation based on the analysis of 5,571 autopsy findings of females under the age of 40 in Japan

Ovarian cryopreservation and autotransplantation could be of potential value for preservation of fertility in the patients with various malignancies. Ovarian tissue should be cryopreserved actively for fertility preservation, but stored tissue should be autotransplanted with much caution until reliable methods are established to detect minimal residual disease in grafts in precise and reproducible manners.

Fertility Preservation: The Rationale for Cryopreservation of the Whole Ovary

Recently, much progress has been made in the area of cryopreservation of ovarian tissue, one of the only options for fertility preservation available to women who require immediate gonadotoxic chemotherapy. Human ovarian cortical tissue strips have been cryopreserved, thawed, and autotransplanted with successful reproductive function. Cryopreservation of ovarian cortical strips, however, is limited by the ischemia that occurs at the time of retransplantation. Thus for patients that desire long-term resumption of endocrine function, cryopreservation of the whole ovary with an intact pedicle and vascular supply may be a better option. This article describes recent advances in whole ovary cryopreservation in both animal and human models, with a focus on surgical technique for removal, choice of cryoprotectants, freezing and thawing protocols, and preliminary results with organ retransplantation. Although no human cases of whole ovary retransplantation after cryopreservation have been performed to date, these preliminary studies have been encouraging, and it is likely that this option for fertility preservation will be a viable treatment option in the future.

Secondary follicle growth and oocyte maturation by culture in alginate hydrogel following cryopreservation of the ovary or individual follicles

An option for fertility preservation for women facing a cancer diagnosis involves the cryopreservation of ovarian tissue for later re-transplantation or in vitro culture, with in vitro culture preferred to avoid reintroduction of the cancer. Small, immature follicles survive the freeze-thaw process, and can be matured through in follicle maturation (IFM) that involves an initial growth of the follicle and subsequent maturation of the oocyte. The ovarian tissue can be cryopreserved in two forms: (i) cortical strips consisting of follicles and surrounding stroma (Cryo-Ov) or (ii) individually isolated follicles (Cryo-In). The aim of this study was to assess the follicle growth and oocyte maturation for follicles that were cryopreserved either as strips or individually using a slow-freezing cryopreservation method. The two follicle groups, together with non-cryopreserved control follicles, were grown in an alginate-based three-dimensional culture system for 12 days. The overall survival, size increase and antrum formation rates were comparable among the three groups. At day 12 of culture, Androstenedione levels were decreased in the Cryo-Ov group relative to the other two, and the ratio of progesterone to estradiol was increased in the two cryopreserved groups relative to the control. Both Gja1 (known as connexin 43) and Gja4 (known as connexin 37) mRNA expression were decreased at day 6 in the cryopreserved groups relative to controls, and by day 12, Gja1 was similar for all three groups. Moreover, Cryo-In resulted in lower GVBD rate indicating some impaired oocyte development. Overall, the present study demonstrated that mouse preantral follicles, either within ovarian tissues or individually isolated, could be successfully cryopreserved by the slow-freezing method, as evidenced by post-thaw follicle development and steroidgenesis, oocyte maturation and molecular markers for oocyte and/or granulosa cells connection.

Development of human preantral follicles in vitro is enhanced by timed exposure to activin-A followed by FSH

OBJECTIVE: The aim of this study was to determine whether exposure to Activin-A followed by FSH would enhance growth and development of isolated human preantral follicles within a recently developed two-step serum free culture system.DESIGN: Ovarian cortical biopsies of approximately 5x4mm were obtained after informed consent during laparoscopic oophorectomy (n=2) and at caesarean section (n=1) from woman aged 40-41 years. Approval for this study was given by the local ethics committee.MATERIALS AND METHODS: Ovarian cortical strips containing primordial and primary follicles were cultured in serum free medium (McCoys + human serum albumen) for 6 days. At the end of this culture period secondary follicles were isolated from the strips. Intact secondary follicles with a mean diameter of 74 +/−5 microns (n = 33) were selected for further culture. Isolated follicles were placed individually within v shaped micro well culture plates in serum free medium in the presence of 100ng/ml of human recombinant activin A. After 2 days 50ng/ml of rhFSH in combination with 100ng/ml of activin A was added to approximately half of the cultured follicles (n=18) whilst the remainder (n=15) were exposed to only activin A for a further 2 days. Follicle diameters were measured on day 2 and day 4, cultured follicles at day 4 were fixed in Bouin's solution and prepared for histology and subsequent morphological analysis to determine oocyte and granulosa cell health.RESULTS: Isolated preantral follicles showed a significant increase in size during the first 2 days of culture in the presence of activin A. No follicular rupture or degeneration was observed during this period. Follicles exposed to FSH after 2 days in vitro showed a significantly higher increase in size (130+/−22 microns) when compared with those cultured in activin alone (91 +/− 13 microns) (p< 0.05). Morphological analysis showed an improvement in follicle health in those follicles exposed to both activin A and FSH.CONCLUSIONS: Primordial follicles can be activated to grow in vitro and develop to preantral follicles within 6 days of culture in cortical strips. Isolated preantral follicles show significant growth during a further 4 day culture period in the presence of activin A. Follicles exposed to a combination of activin A and FSH show a significantly greater rate of growth and improved morphology compared to activin alone. These preliminary results represent further progress towards achieving full in vitro growth of human oocytes. OBJECTIVE: The aim of this study was to determine whether exposure to Activin-A followed by FSH would enhance growth and development of isolated human preantral follicles within a recently developed two-step serum free culture system. DESIGN: Ovarian cortical biopsies of approximately 5x4mm were obtained after informed consent during laparoscopic oophorectomy (n=2) and at caesarean section (n=1) from woman aged 40-41 years. Approval for this study was given by the local ethics committee. MATERIALS AND METHODS: Ovarian cortical strips containing primordial and primary follicles were cultured in serum free medium (McCoys + human serum albumen) for 6 days. At the end of this culture period secondary follicles were isolated from the strips. Intact secondary follicles with a mean diameter of 74 +/−5 microns (n = 33) were selected for further culture. Isolated follicles were placed individually within v shaped micro well culture plates in serum free medium in the presence of 100ng/ml of human recombinant activin A. After 2 days 50ng/ml of rhFSH in combination with 100ng/ml of activin A was added to approximately half of the cultured follicles (n=18) whilst the remainder (n=15) were exposed to only activin A for a further 2 days. Follicle diameters were measured on day 2 and day 4, cultured follicles at day 4 were fixed in Bouin's solution and prepared for histology and subsequent morphological analysis to determine oocyte and granulosa cell health. RESULTS: Isolated preantral follicles showed a significant increase in size during the first 2 days of culture in the presence of activin A. No follicular rupture or degeneration was observed during this period. Follicles exposed to FSH after 2 days in vitro showed a significantly higher increase in size (130+/−22 microns) when compared with those cultured in activin alone (91 +/− 13 microns) (p< 0.05). Morphological analysis showed an improvement in follicle health in those follicles exposed to both activin A and FSH. CONCLUSIONS: Primordial follicles can be activated to grow in vitro and develop to preantral follicles within 6 days of culture in cortical strips. Isolated preantral follicles show significant growth during a further 4 day culture period in the presence of activin A. Follicles exposed to a combination of activin A and FSH show a significantly greater rate of growth and improved morphology compared to activin alone. These preliminary results represent further progress towards achieving full in vitro growth of human oocytes.

Pregnancy following homologous prepubertal ovarian transplantation in the dog

In several canine models of hereditary human disease the homozygote dogs die prior to puberty, or have substantially reduced fertility. To create a clinically healthy animal that can be bred, but can also transmit the gene of interest, a model of homologous ovarian transplantation in prepubertal dogs was developed. Six dog leukocyte antigen (DLA) identical littermates underwent transplantation of ovarian cortical strips (n = 2) or the entire ovary (n = 4). Immunosuppression was maintained with cyclosporine and MMF in the immediate post-operative period and cyclosporine alone thereafter. All 6 dogs entered puberty and normal semiannual estrus cycles as demonstrated by both physical changes and increasing serum progesterone. Four dogs were bred to a proven stud male, and one became pregnant. Three viable fetuses with observable heartbeats were detected on ultrasound examination. Although the dog eventually aborted the litter, this work represents the first pregnancy achieved following a prepubertal ovarian transplant in the dog.

Selective impairment of the cerebellar C1 module involved in rat hind limb control reduces step-dependent modulation of cutaneous reflexes.

The cerebellum is divided into multiple parasagittally organized modules, which are thought to represent functional entities. How individual modules participate in cerebellar control of complex movements such as locomotion remains largely unknown. To a large extent, this is caused by the inability to study the contribution of individual modules during locomotion. Because of the architecture of modules, based on narrow, elongated cortical strips that may be discontinuous in the rostrocaudal direction, lesion of a complete module, without affecting neighboring modules, has not been possible. Here, we report on a new method for inducing a selective dysfunction of spatially separated parts of a single module using a small cortical injection of a retrogradely transported neurotoxin, cholera toxin b-subunit-saporin. We show that such a local injection into the C1 module results in climbing fiber and partial mossy fiber deafferentation of functionally related areas of this module, thereby resulting in a severe impairment of the whole module without affecting neighboring modules. A subsequent functional analysis indicates that such an impairment of the hindlimb part of the C1 module did not have a significant impact on skilled walking or overall stepping pattern. However, the modulation of cutaneously induced reflexes during stepping was severely diminished. We propose that the C1 module is specifically involved in the adaptive control of reflexes.

Cryopreservation of intact human ovary with its vascular pedicle

The aim of this study was to assess the immediate post-thawing injury to the human ovary that was cryopreserved either as a whole with its vascular pedicle or as ovarian cortical strips. Bilateral oophorectomy was performed in two women (46 and 44 years old) undergoing vaginal hysterectomy and laparoscopic hysterectomy, respectively. Both women agreed to donate their ovaries for experimental research. In both patients, one of the harvested ovaries was sectioned and cryopreserved (by slow freezing) as ovarian cortical strips of 1.0 x 1.0 x 5.0 mm(3) each. The other ovary was cryopreserved intact with its vascular pedicle. After thawing 7 days later, follicular viability, histology, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labelling (TUNEL) assay (to detect apoptosis) and immunoperoxidase staining (to define Bcl-2 and p53 protein expression profiles) of the ovarian tissue were performed. Tissues from non-cryopreserved ovaries served as control specimens (two cases). The overall viability of the primordial follicles was 75 and 78% in intact cryopreserved-thawed (C-T) ovaries and 81 and 83% in ovarian cortical strips in the 46- and 44-year-old patients, respectively. Comparable primordial follicle counts, absence of features of necrosis, mean values of apoptosis and weak Bcl-2 and p53 protein expressions were observed both in the intact C-T ovary and in the C-T ovarian cortical strips. Cryoperfusion and cryopreservation of entire human ovary can be achieved with the maintenance of excellent viability of the superficial and the deeper tissues using a slow-freezing protocol. Cryopreservation injury is associated neither with significant alteration in the expression pattern of Bcl-2 and p53 proteins in the ovarian tissues nor with significant follicular damage.

P-474: Assessment of follicular viability, apoptotic markers and blood vessels status after cryopreservation of intact human ovary with its vascular pedicle

The aim of the current study is to assess the immediate post thawing injury to the human ovary that was either cryopreserved as a whole with its vascular pedicle or as ovarian cortical strips. Experimental study. Bilateral oophrectomy was performed to two women (46 and 44 years old) undergoing vaginal hysterectomy and laparoscopic hysterectomy, respectively. Both women agreed to donate their ovaries for experimental research. In both patients, one of the harvested ovaries was sectioned and cryopreserved as ovarian cortical strips of 1.0 x 1.0 x 5.0 mm3, each. The other ovary was cryopreserved intact with its vascular pedicle and subsequently thawed 7 days later. After thawing, follicular viability, histology, TUNEL assay (to detect apoptosis) and immunoperoxidase staining (to define bcl-2 and p53 protein expression profile) of the ovarian tissue were performed. Tissues from two non-cryopreserved ovaries from age matched patients served as control specimens. The overall viability of the primordial follicles was 75% and 78% [intact cryopreserved-thawed (C-T) ovaries] and 81 and 83% (ovarian cortical strips) in the 2 patients respectively. Comparable primordial follicle counts, absence of features of necrosis, mean values of apoptosis and weak bcl-2 and p53 protein expression were observed both in the intact C-T ovary and C-T ovarian cortical strips. There were no significant differences between cryopreserved-thawed ovarian tissue and controls regarding the vascular density (Table).Tabled 1 Follicualr viability, apoptotic markers and postvascualr density are comparable between cryopreserved-thawed ovarian and ccontrols. Cryopreservation injury is associated neither with significant alteration in the expression pattern of bcl-2 and p53 proteins in the ovarian tissues nor with significant follicular damage.

P-408: Evidence for altered ovarian stromal function in women previously exposed to chemotherapy

While numerous chemotherapy agents are known to cause ovarian follicle cell death, it is unknown whether chemotherapy has a detrimental effect on ovarian stromal cells. Our purpose was to ascertain whether the stromal function is altered in patients who had received chemo- and/or pelvic radiotherapy compared to those who did not receive previous treatment, as determined by in vitro estradiol production. Because ovarian cortical strips mostly contain primordial and primary follicles, in vitro production of estradiol would reflect stromal cell function.

Two novel techniques to detect follicles in human ovarian cortical tissue

Ovarian tissue cryopreservation and transplantation are becoming increasingly important issues for preserving female fertility as shown by recent successes in restoring ovarian activity and even fertility. Primordial follicle content before transplantation is a key issue for success. We investigated two novel methods to detect primordial follicles in human ovarian cortical tissue strips. The first method used the fluorescent mitochondrial stain rhodamine 123 in combination with laser scanning confocal microscopy (LSCM). The first method used the fluorescent mitochondrial stain rhodamine 123 (R123) in combination with laser scanning confocal microscopy (LSCM). The second used a simple stereomicroscopic method with glass-bottom dishes for detecting primordial follicles in ovarian cortical tissue strips. Potential toxic effects of R123 and of the exposure to confocal laser were investigated in a mouse ovarian allograft model. Follicles were visible as white spots in thin cortical strips using LSCM in single and fast scanning at low magnification, allowing a fair estimation of the number of primordial follicles present. Using the second method, ovarian follicles were also visible using glass-bottom dishes under the stereomicroscope, although tissue thickness and density were limiting factors of its success. Follicles can be visualized in human cortical ovarian strips with R123 in combination with LSCM. Stereomicroscopy using glass-bottom dishes and transmitted illumination is a simple alternative method and has the advantage of allowing further safe clinical use of the analysed tissue.

Ovarian Cortical Transplantation

Evaluation of: Kiran G, Kiran H, Coban YK, Guven AM: Ovarian cortical transplantation may be an alternative to hormone therapy in patients with early climacterium. Fertil. Steril. 84, 1509 (2005) [1] . Describes a report of a 46-year-old woman who had a total abdominal hysterectomy and bilateral salpingo-oophorectomy. After surgery, the patient underwent ovarian cortical transplantation of fresh autologous ovarian cortical strips to her lower abdomen. Follicle-stimulating hormone, luteinizing hormone and estradiol were measured at 3-month intervals. This paper describes the last year of her sampling, ending at 18 months postsurgery. The patient had no menopausal symptoms. Hormonal secretion was at times at postmenopausal levels and at times at premenopausal levels. There was evidence of folliculogenesis and ovulation.

Contribution of Transmission Electron Microscopy To the Study of Human Ovarian Tissue Integrity After Enzymatic Isolation, Cryopreservation or Xenografting

Many therapies indicated for the treatment of malignancy in female cancer patients unfortunately carry a severe risk of subsequent infertility. Preservation of the follicular reserve before gonadotoxic treatment can be studied on three different levels: isolated follicles, follicles embedded in ovarian cortical strips or in an intact ovary. For this purpose, enzymatic isolation, intact ovary cryopreservation and xenografting are different treatments currently under investigation in our laboratories. Our aim is to evaluate by transmission electron microscopy (TEM) the impact of enzymatic isolation, cryopreservation or xenografting on the tissue and cell integrity of human ovarian tissue. Prospective experimental study. Ovarian tissue was analyzed by TEM after the following treatments: Treatment 1- Enzymatic isolation: eight human ovarian biopsies were processed for follicle isolation by collagenase or Liberase enzymatic digestion. One hundred and nine isolated human primordial/primary follicles were fixed and processed for TEM. Treatment 2- Intact ovary cryopreservation : three human ovaries were frozen with their vascular pedicle using a passive cooling device. At three different steps (freshly removed ovary, after perfusion with cryoprotectant and after thawing), samples were taken and processed for TEM. Treatment 3- Xenografting of ovarian cortical strips: sixteen human ovarian cortical biopsies were frozen-thawed (FT). Eight FT biopsies were grafted in the peritoneum of nude mice and removed after 3 weeks. At three different steps (fresh, FT and grafted tissue), samples were taken and processed for TEM. In treatment 1, the majority of isolated follicles were well preserved, but surrounded by a discontinous basal lamina. Numerous connective tissue fibres were found on the outer aspect of the residual basal lamina. Some follicles showed ultrastructural signs of atresia, such as patches of heterochromatin, focal discontinuities at the oolemma-follicular cell interface, irregularities in the nuclear profile, clusters of lipid droplets or small vacuoles and myelin-like structures. In treatment 2, we found a well preserved follicular ultrastructure, stromal and vascular compartments after freezing and thawing. Occasionally, ultrastructural signs of atresia were observed in some follicles (as described above). In treatment 3, healthy-looking and altered primordial and primary follicles were found in the 3 groups. Altered follicles showed oocyte vacuolization and/or fragmentation, degeneration of follicular cells and increased thickness of the basement membrane. A few growing preantral follicles observed after xenografting contained intact oocytes, in which organelles showed a perinuclear distribution typical of earlier developmental stages. We evaluated the degree of maturity of neo-vessels in xenografts. TEM allows enhanced evaluation of actual cell structure, providing some accurate information, hardly obtained by histology or other analysis. We could evidence early signs of atresia in all cellular compartments, such as swelling of the mitochondria; alterations in follicular basal membrane and follicular cell-oocyte interactions, crucial for follicular maturation; or integrity and maturity of vessels. Morpho-functional analysis by TEM allows a marked improvement in the evaluation of integrity of human ovarian tissue.

Restoration of ovarian function after orthotopic (intraovarian and periovarian) transplantation of cryopreserved ovarian tissue in a woman treated by bone marrow transplantation for sickle cell anaemia: Case report

Ovarian function after orthotopic transplantation of cryopreserved ovarian tissue has been restored in women with malignant disease. Here the techniques are adapted for a non-cancer patient. In 1999, right oophorectomy was performed in a 21 year old woman before chemotherapy, prior to bone marrow transplantation. Ovarian cortex was frozen, according to a strict protocol. After thawing, ovarian cortex was reimplanted into the ovary and in a peritoneal window close to the ovary in 2004. Four-and-a-half months after reimplantation, LH, FSH, 17beta-estradiol and progesterone levels, as well as ultrasonography, demonstrated the presence of an ovulatory cycle. After this cycle, the patient experienced two other ovulatory cycles, evidenced by FSH and 17beta-estradiol concentrations, as well as ultrasound demonstration of a follicle. Follicular development was clearly observed in both the intraovarian site (1st and 2nd cycle) and the peritoneal window (3rd cycle). Restoration of endocrine ovarian function occurred after ovarian cortical strips, biopsied and cryopreserved before chemotherapy, were reimplanted into the ovary itself and a periovarian peritoneal window.

Fresh autologous transplantation of ovarian cortical strips to the anterior abdominal wall at the pfannenstiel incision site

To describe an open surgical technique for transplanting fresh ovarion tissue to the anterior abdominal wall at the incision site and to determine whether ovarian function would be restored after transplantation. Case study. Academic medical center. A 44-year-old patient who underwent an operation for uterine fibroids. Microsurgical reconstruction of ovarian cortex and its transplantation to the anterior abdominal wall at incision site of Pfannenstiel. Follicular development evident by ultrasound examination; restoration of serum FSH and LH levels to nonmenopausal range; and disappearance of menopausal symptoms. Early postoperative FSH, LH, and E(2) levels showed that menopause was confirmed. Postoperative hormone levels at months 2, 3, and 6 were as follows: FSH: 77.86, 79.50, and 13.70 mIU/mL; LH: 34.60, 33.92, and 8.78 mIU/mL; E(2): 29, 46, and 48 pg/mL. The patient is still followed-up for postmenopausal status. Autotransplantation of cortical strips to the anterior abdominal wall at the incision site without further incision can be a logical solution for the patients at early climacteric age.

Long-term ovarian function in sheep after ovariectomy and autotransplantation of cryopreserved cortical strips.

Transplantation of cryopreserved ovarian tissue offers a means of preserving reproductive function in girls/women whose ovarian function is compromised by radio- and/or chemotherapy for cancer. Studies in small laboratory rodents have demonstrated that fertility can be restored by transplanting whole cryopreserved ovaries. In large animals, it is not possible to freeze or transplant ovaries without vascular anastomosis. However, primordial oocytes in cortical strips survive freezing, thawing and culture in vitro. We have used sheep as an experimental model for the human because the size of the ovary and length of folliculogenesis (4-6 months) are similar. Following orthotopic transplantation the grafts, become vascularised within a few days and ovulatory cycles become established by 4 months. Several lambs have been born in ewes after autotransplantation of the grafts which continue to function for up to 2 years. The levels of FSH remain consistently raised after transplantation due to a reduced secretion of inhibin A, reflecting the reduction in the size of the ovarian pool of follicles.

A technique for transplantation of ovarian cortical strips to the forearm

Objective To describe a forearm heterotopic ovarian transplantation technique. Design Case study. Setting Academic medical center. Patient(s) One patient with stage IIIB squamous cell cervical carcinoma and one patient with recurrent benign ovarian cysts. Intervention(s) Preparation of thin ovarian cortical slices and transplantation under the skin of the forearm. Main outcome measure(s) Follicular development and oocyte retrieval; cyclical estradiol (E 2) and progesterone (P 4) production; restoration of serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels to reproductive age range. Result(s) Both patients were menopausal immediately after oophorectomy. The first patient developed a dominant follicle 10 weeks after transplantation, and her gonadotropin levels decreased to nonmenopausal levels. Percutaneous aspiration of ovarian follicles yielded a metaphase I (M-I) oocyte that was matured to metaphase II (M-II). The first patient’s graft was functional for at least 21 months. In the second patient, ovarian follicle development was detected 6 months after transplantation, and periodic menstruation occurred thereafter. Spontaneous ovulation was confirmed by a midluteal increase in her P 4 levels. Menstruation and follicle development continued for more than 2 years after the transplant. Conclusion(s) Heterotopic transplantation of ovarian tissue to the forearm is a simple and promising technique to restore ovarian function in women who become menopausal due to chemotherapy, surgery, or radiation.

Assessment of tissue injury in cryopreserved ovarian tissue

Assessment of tissue injury in cryopreserved ovarian tissue

Restoration of ovarian function after autotransplantation of intact frozen-thawed sheep ovaries with microvascular anastomosis

Objective To test the feasibility of transplanting an intact frozen-thawed ovary with microvascular anastomosis of the ovarian vascular pedicle to the deep inferior epigastric vessels. Design Chronic survival study. Setting Biological Resources Unit, The Cleveland Clinic Foundation. Animal(s) Adult merino ewes. Intervention(s) Bilateral laparoscopic oophorectomy was performed on 17 synchronized ewes. In one group of animals (Group I, n = 11), both ovaries were cryopreserved intact with their vascular pedicles. In another group of animals (Group II, n = 6), ovarian cortical strips were prepared from each ovary and cryopreserved. After thawing, follicular viability and apoptosis rates were assessed using one ovary. The other ovary was transplanted to the abdominal wall with microvascular anastomosis (Group I). In Group II, the ovarian cortical strips were placed in the anterior abdominal wall. Ovaries were harvested after 8–10 days in situ and subjected to histological evaluation. Main outcome measure(s) Blood flow, apoptotic signals, follicular viability, serum estradiol (E 2), follicle-stimulating hormone (FSH), and histology. Result(s) No significant differences were found in the mean values of apoptosis (mostly in the atretic and some secondary follicles) and follicular viability in both groups. In Group I, immediate and long-term patency were documented in 100% and 27% (3/11) of the grafts, respectively; and postoperative FSH levels were similar to preoperative values in animals with patent vessels. In Group II, postoperative FSH levels were significantly higher than the preoperative ones ( P=.03). Conclusion(s) Transplantation of an intact frozen-thawed ovary is technically feasible. Using this approach, immediate restoration of vascular supply and ovarian hormonal functions is possible.