Meeting abstracts

How to provide a good vascularisation on ovarian tissue grafting and pregnancies.

Piver,P*; Leperlier,F*; Pech,j.c*; Roux,C**; Paulhac,s*; Gauthier,t*; Maubon*** A, Vidal**** E, Aubard,y*,*IVF center , ***radiology dept, ****medicine dept ;university hospital Limoges France.**IVF center university hospital Besancon, France.

Since 1995, in France, about 700 children or women have had their ovary cryopreserved for fertility preservation before castrative treatment. In the world, only 5 births have been published after auto- grafting of ovarian cryopreserved tissue. One of the limitations of the technique is the low survival rate of the follicles after grafting. These patients are usually poor responders after ovarian stimulation. The survival rate of the follicles may depend on the quality and the delay of neovascularisation.Dissen in 1994 and Long in 2007 demonstrated that VEGF secretion of ovarian grafts was maximum at 3 to seven days after the grafting. In 1999, a 27 year old woman presented Systemic Vasculitides. She received 3 bolus of 0.850 g of cyclophosphamide (endoxan), before right ovarian cryopreservation was performed with a slow cooling protocol. During the next two years, the patient received cyclophosphamide (7,65 g IV and 58 g per os). In 2003 she presented a clinical and biological menopause, with high levels of Fsh and Lh. HRT was prescribed during 4 years. In 2007, she wished to be pregnant and asked for her ovarian tissue.The transplantation surgical procedure was the following: During a first laparoscopy, one large fragment of ovarian cortex was rapidly thawed and cut in 10 pieces of 1 to 2 millimetres. We opened a left peritoneal window (LPW) near the uterine vessels, and small pieces of the ovarian cortex were put there. Other small pieces were put in an ovarian window made in the left remaining ovary (LO) by a large biopsy.A second laparoscopy was performed 3 days later, during the VEGF secretion rise. 8 large fragments of 8 mm² were grafted: 2 on the LO, 3 on the LPW, and 3 on a new peritoneal window (RPW) near the right uterus vessels. A total of 4 IVF were performed after ovarian stimulation 23 ooocytes, 17 M II, 16 embryos, 7 blastocysts and 2 pregnancies were obtained one ectopic and a second ongoing in its third trimester . The graft on the LPW provided 80% of the oocytes harvested and 90% of the mature oocytes and embryo. All the embryos transferred and both pregnancies came from the LPW graft. Doppler and Angiography MRI located the origin of the neovascularisation of the LPW on the ombilico-uterin artery. The new vessels are larger than 1 mm.We report new pregnancies after cryopreservation of ovarian tissue and auto-grafting, with a new technique using a small piece of ovarian tissue to stimulate angiogenesis factor, providing good neovascularisation for grafting 3 days later. We obtained a large number of oocytes, blastocysts, embryos, and 2 pregnancies in only 4 cycles. One is an ongoing pregnancy in its third trimester. This is the first time that a pregnancy is obtained with ovarian tissue that was frozen for 8 and a half year.

Autotransplantation of cryopreserved ovarian tissue in a cohort of 12 Danish women

Kirsten Tryde Schmidt^1,2, Mikkel Rosendahl², Erik Ernst³, Anders Nyboe Andersen¹, Anne Loft¹ and Claus Yding Andersen². ¹The Fertility Clinic and ²The Laboratory of Reproductive Biology, University Hospital of Copenhagen, Rigshospitalet, Denmark, ³The Fertility Clinic, Skejby University Hospital, Denmark.

Antineoplastic treatments have a known gonadotoxic effect, which in women can cause premature ovarian failure (POF) and unwanted infertility. Cryopreservation of ovarian tissue has been developed as a means to restore ovarian function and possibly fertility in these women. We report our experience in a cohort of 12 Danish women who had some of their cryopreserved ovarian tissue re-implanted after treatment-induced POF. Patients and methods: Among the 12 women, 9 had a malignant and 3 a benign disease. The mean age at cryopreservation was 28.4 years. All had a potentially gonadotoxic treatment between March 2000 and July 2006 and cortical tissue from one ovary cryopreserved prior to treatment. All developed amenorrhea and hot flushes after treatment and POF was confirmed by ultrasonography of the remaining ovary and by menopausal levels of FSH values (mean 74 IU/l). Autotransplantation was performed between 17 and 65 months after end of treatment. Five of the patients returned for a second autotransplantation due to cessation of function in the first transplant or to increase chances of pregnancy. The ovarian tissue was transplanted to both orthotopic and heterotopic sites. Results: All women regained menstrual cycles within 8–26 weeks (mean 19 weeks) after transplantation, levels of FSH and LH were normalised, oestradiol levels increased and antral follicles appeared on ultrasonography. Ten of the women underwent a total of 43 in vitro fertilisation (IVF) cycles, 35 oocytes were aspirated, 21 were fertilised and 14 embryos were transferred resulting in five pregnancies: two biochemical, one clinical that miscarried in week seven and two ongoing that resulted in the delivery of two healthy babies born at term to two women. One of these women subsequently conceived spontaneously, which resulted in the delivery of another healthy baby. : Autotransplantation of cryopreserved ovarian tissue has proven to be an efficient way of restoring ovarian function in women with treatment-induced POF. In our material all women regained ovarian function and four women additionally became pregnant, after IVF or spontaneously, resulting in the delivery of three children to two women. However, IVF in this subgroup of infertility patients is difficult and raises many challenges.

Ovarian cortex cryopreservation in girls under 16 years of age

Jadoul P, Donnez J. Cliniques Universtitaires Saint-Luc, UCL, Brussels, Belgium

In prepubertal and adolescent girls, fertility may be impaired by gonadotoxic treatments, repeat ovarian surgery or genetic disorders. Cryopreservation of ovarian cortex is an existing option to preserve fertility, and pregnancies have been obtained after reimplantation of frozen-thawed ovarian cortex in adults. Our aim was to evaluate the feasibility and safety of ovarian cortex cryopreservation in young girls at risk of premature ovarian failure. We reviewed data from 58 girls under 16 years of age, who underwent ovarian tissue harvesting and cryopreservation in our institution between May 2001 and May 2009. Patient age ranged from 10 months to 15 years (10.4 ± 4.4 years). Twenty-one girls were under 10 years of age and 38 were prepubertal. Forty-eight patients were suffering from cancer, three from genetic disorders, one from systemic disease requiring chemotherapy, and four from benign ovarian pathologies. Two patients required cryopreservation prior to bone marrow transplantation for benign disease. We analyzed the feasibility and safety of the procedure, and reviewed our indications. All procedures were performed by laparoscopy. Unilateral or bilateral cortical biopsies were taken from 38 patients, and unilateral oophorectomy was performed in 20 patients. No complications occurred during surgery. Histological analysis of a small piece of ovarian cortex showed the presence of follicles in all cases, except one patient suffering from galactosemia. In one patient with lymphoma, laparoscopy revealed tumoral infiltration of the ovary by a 10-cm mass, confirmed by histological analysis. No ovarian involvement was observed in any other cases. For all oncological indications, chemotherapy was initiated from day 0 to day 5 after cryopreservation. During subsequent months, seven girls (12%) changed categories from low or medium risk to high risk of premature ovarian failure.Our series shows that ovarian tissue cryopreservation is feasible at any age by a laparoscopic approach, without complications and without postponing cancer treatment. We also demonstrate that it is virtually impossible to give the patient or her parents an accurate assessment of the risk to fertility. Although we can define treatment regimens associated with varying degrees of risk, disease evolution is never completely predictable. We therefore believe that fertility preservation options should be discussed with all patients at risk of impaired fertility, even very young girls.

Cryopreservation of ovarian tissue for fertility preservation in girls.

Kirsten Tryde Schmidt^1,2, Katharina Main³, Sara Markholt⁴, Emil H. Ernst⁴, Anders Nyboe Andersen¹, Erik Ernst⁴, Mikkel Rosendahl² and Claus Yding Andersen^{2. 1}The Fertility Clinic and ²The Laboratory of Reproductive Biology, University Hospital of Copenhagen, Rigshospitalet, ³The Department of Growth and Reproduction, University Hospital of Copenhagen, Rigshospitalet, ⁴Reproductive Laboratory, Institute of Anatomy, University of Aarhus.

Chemo- or radiation therapy treatment in girls may cause unwanted long-term side effects such as absent menarche or early onset of premature ovarian failure (POF) after puberty. Cryopreservation of ovarian tissue prior to gonadotoxic treatment can be offered as a means of preserving fertility in some of these patients. This study describes a cohort of 88 Danish girls with cryopreserved ovarian tissue, which is probably the largest series of its nature available. From 2000 to 2009 a total of 88 girls <18 years of age had one entire ovary removed and cryopreserved at Rigshospitalet, Copenhagen before bonemarrow transplantation (BMT) or treatment with high-dose alkylating agents for a malignant (N = 69) or benign (N=15) disease. Additionally, four girls had one ovary cryopreserved at a young age where follicles were still expected to be present (Turner mocaisism and galactosaemia). A small piece of ovarian tissue was assessed histologically and the follicular density calculated. After treatment a pediatrician examined the girls at regular intervals for pubertal status and ovarian function by means of Tanner staging, menstrual history and measurements of FSH and oestradiol. So far, we have data on pubertal status after treatment on 22 girls >10 years of age: 13 showed signs of ovarian failure (OF) with elevated FSH values >20 IU/l (mean 71 IU/l) and absent menarche or amenorrhea, seven had spontaneous menstruations and in two girls it was too early to assess ovarian function. Nine of the girls with OF had been treated with BMT preconditioned with total body irradiation. Puberty was induced in two girls and hormonal replacement therapy given to 12 of the girls with OF. Data on ovarian function in an additional 24 girls will be presented as well as data on follicular density. As of today, a total of 21 girls in the programme have died from their disease. Of the girls so far evaluated for ovarian function the majority (59%) had OF after treatment. Although still experimental, cryopreservation should be considered in girls at high risk of OF, since no other options for fertility preservation exist in these young patients.

Oncologists’ practice and attitudes regarding fertility preservation in female cancer patients: a pilot study in the Netherlands.

L.A. Louwé¹, M.M. ter Kuile¹, E. Jenninga¹, L.M. Tiemessen¹, C.G.J.M. Hilders^2, J.W.R. Nortier³, A.A.W. Peters^1, A.M. Stiggelbout^{4 1}Department of Gynaecology, ³Department of Medical Oncology , ⁴Department of Medical Decision Making, Leiden University Medical Center, Leiden, the Netherlands;² Department of Gynaecology, Reinier de Graaf Hospital, Delft, the Netherlands

Fertility changes are a common side effect of cancer treatment. The combination of improved survival and advances in reproductive medicine provide some hope for fertility preservation after cancer treatment in women. The aim of this study was to assess oncologists’ practice and attitudes regarding treatment-related infertility and regarding fertility preservation. Recruitment letters were mailed to a selected group of 454 oncologists; the letter referred to a link to complete a 7-item questionnaire online. Hundred and eighty-eight of the 454 oncologists (41%) responded. Ninety-six of the 188 (51 %) oncologists saw at least 5 women with cancer within the reproductive age group, during the last year. These 96 questionnaires were used for analysis. The sample included 28 (29%) gynaecologists, 22 (23%) medical oncologists, 19 (20%) surgeons, 16 (17%) radiotherapists and 11 (12%) haematologists. Mean number of years of practice was 13.3 years; seventy percent of the sample was male. Nearly 70% of the oncologists reported that at least one of their patients had undergone fertility preservation prior to cancer treatment. The most important reason for not offering fertility preservation was “factors concerning the disease” (61%). About one-third of the oncologists (31.3 %) did not discuss fertility issues. If the possible fertility preservation options were standardized almost half of the oncologists would refer 75–100% of their patients for fertility preservation. No significant differences were found between oncologists in (1) the mean value of raising fertility issues with premenopausal women prior to cancer treatment (5.9 on a 7-point scale) and (2) the mean value of fertility after a malignant disease (5.6 on a 7-point scale). Early results indicate that most of the oncologists are willing to discuss fertility issues with premenopausal women prior to the cancer treatment, but currently this is not done by one-third of the oncologists.

Systematic follow-up of young patients undergoing chemotherapy in order to assess the dynamics of follicular depletion: what should we learn? The experience of the Lille University Hospital.

Decanter C, Morschhauser F, Lefebvre C, Leroy-Martin B, Gallo C, Pigny P, Dewailly D. Hôpital Jeanne de Flandre, Lille, France.

Chemotherapy protocols are ovariotoxic by damaging all follicles. To better characterise the degree and the evolution of follicular depletion in patients treated for lymphoma, we used serial and repeated anti-Müllerian hormone (AMH) measurements and antral follicle counts (AFC). 45 young women were prospectively recruited before the initiation of chemotherapy for Hodgkin’s or non-Hodgkin’s lymphoma. They were assigned either to an ABVD protocol (ABVD group) or to a protocol that included more ovariotoxic drugs such as cyclophosphamid (non-ABVD group). Serum samples for AMH assays and AFC were performed before and during chemotherapy and every three months after the end of treatment for a period of two years. Mean AMH levels fell drastically and rapidly after the start of chemotherapy in a similar fashion between both groups. However, the ovarian recovery phase was significantly different between the 2 groups with an AMH re-increase significantly more pronounced in the ABVD group returning to pre-treatment values twelve months after the end of treatment. Conversely, in the non-ABVD group, AMH values after chemotherapy remained very low or undetectable. The dynamics of the AFC showed the same pattern than AMH in both groups. Longitudinal study of AMH levels and AFC during and after chemotherapy well reflects the dynamics of follicular depletion. It highlights differences between protocols that could help in the understanding of the ovarian toxicity and, ultimately, in fertility preservation counselling.

Microarray approach to investigate gene expression in human ovarian tissue after xenografting.

Romeu L. and Van Langendonckt A. Gynecology Research Unit (J.Donnez), Université Catholique de Louvain, Brussels, Belgium

A massive activation of follicular growth and extensive stromal tissue fibrosis is observed after xenotransplantation of cryopreserved human ovarian tissue. This premature follicular activation may be a result of a deregulation of folliculogenesis or a stimulation of follicular cell growth caused by ischemic stress. Our aim was to investigate the impact of avascular human ovarian cortex xenografting on gene expression in order to better understand the mechanisms involved. Nude mice were grafted intraperitoneally with frozen-thawed human ovarian tissue from 8 patients. Grafts were recovered after 2 (D2) or 7 (D7) days. Non-grafted tissue was considered as control (D0). Total RNA was extracted using phase separation (Tripure) and its quality was analyzed. Inclusion criterion was RIN ≥ 6.5 (RNA integrity number). Differentially expressed transcripts between D0, D2 and D7 (n = 4 biopsies) were identified by Human Genome U133 Plus 2.0 Array (Affymetrix) using a linear analysis model. Main function and gene interactions were highlighted with Ingenuity Pathways Analysis (IPA) software. Transcripts showing a fold change >2 or <0.5 and p-value <0.01 were considered to be up- or down-regulated. Taqman RT-PCR with 7 primer/probe sets was used to validate array results (n = 8 biopsies). Microarray analysis revealed 99 and 326 up-regulated, 321 and 705 down-regulated transcripts on D2 and D7 respectively compared to D0. IPA identified factors linking hypoxic response, inflammation and revascularization. Molecules involved in hypoxia response (TGFB1, MMP14) and inflammation (IL8) stimulate the production of mediators of both angiogenesis (VEGF, PlGF) and follicular development (inhibins). Moreover, STAT1 and ER1, exerting a negative control of cell cycle, are down-regulated while LIF (proliferation inducer) is up-regulated after ovarian tissue grafting. These three molecules may be implied in the activation of follicular proliferation. Our study identified, for the first time, the pathways involved in apoptotic and inflammatory processes that are linked to revascularization of human ovarian tissue after grafting. MMP14, IL8 and PlGF involved in these pathways are promising targets to enhance revascularization processes. Over-expression of these molecules may have an implication in the deregulation and subsequent loss of follicles.

In vitro maturation and vitrification of immature oocytes combined with ovarian tissue cryopreservation: a new strategy of fertility preservation.

Fasano, G.^{a, b}; Demeestere, I.^{a, b}; Moffa, F.^b; Dechène, J.^a; Tsepelidis S.^a,b; Bockstaele L.^a and Englert, Y^a,b.

^aResearch Laboratory on Human Reproduction, Faculty of Medicine, Campus Erasme, Université Libre de Bruxelles, Belgium

^bFertility Clinic, Department of Obstetrics and Gynecology, Hôpital Erasme, Université Libre de Bruxelles, Belgium

A new attractive combined option to preserve fertility is to vitrify in vitro matured oocytes isolated at the time of ovarian tissue cryopreservation to improve future reproductive potential.

From November 2008 to July 2009, 16/21 patients between 8 and 36 years old underwent the combined procedure. Oocytes-cumulus complexes (OCCs) were retrieved from fresh ovarian cortex after punction of antral follicles and filtration (Falcon, Cell Strainer 352350, 70 µm Nylon) of discarded material. The OCCs were washed and incubated at 37°C in 5% CO₂ in a commercial medium (Sage, IVM Kit media) supplemented with 75 mIU/ml FSH and 75 mIU/ml LH. After 24 h, the OCCs were denuded and metaphase II (MII) oocytes were vitrified. Remaining immature oocytes were kept in IVM media for additional 24 h. All oocytes were vitrified following a standard protocol (Irvine, Vitrification Kit media) using aseptic devices (CryoBiosystem, VHS Kit). 68 oocytes, 52 germinal vesicles (GV) and 16 metaphases I (MI), were isolated and subjected to IVM. 19 MII oocytes were obtained and vitrified (27.94%), 14 and 5 MII after 24 h and 48 h respectively. In 4/16 patients, no oocytes were found (25%). Interestingly, we were able to collect and in vitro mature oocytes regardless to menstrual cycle phase or hormonal treatment, while better results were obtained for patients using hormonal treatment (IVM rate: 36.36% vs 23.91% when no hormonal treatment was taken) . In one patient 10,000 IU of hCG was administered, 13 GV were retrieved and 5 MII were vitrified. Our preliminary experience demonstrates that immature oocytes can be retrieved from the excised ovarian tissue, matured in vitro and vitrified. Combination of both techniques could be an option to improve results of the fertility preservation program. It may be the only option when the risk of auto-transplantation is too high. Oral contraception and hCG priming before collection may increase recovery and maturation rate. Nevertheless, this strategy should currently be regarded as still experimental.

Localization of c-kit/kit ligand and anti-Müllerian hormone in ovarian follicles following 28 weeks of human ovarian tissue xenotransplantation

David A; Amorim CA

Gynecology Research Unit, Université Catholique de Louvain, Brussels, Belgium

Ovarian tissue cryopreservation and transplantation constitute a promising alternative for safeguarding fertility in cancer patients. However, these procedures influence preantral follicle survival. This may be due to alterations in the expression of important growth and/or inhibitory factors, such as c-kit, kit ligand (KL) and anti-Müllerian hormone (AMH). Our aim was to investigate the effects of freezing and grafting on the expression of these factors in human follicles. Ovarian biopsies from 8 patients were used for fresh and frozen-thawed xenografting to 13 SCID mice in the intraperitoneal site for a period of 28 weeks, including 2 weeks of gonadotropin stimulation. In the 4 treatment protocols (fresh and frozen-thawed grafted and non-grafted ovarian tissue), expression of c-kit, KL, and AMH were assessed by immunohistochemistry, and follicular proportion, follicular diameters were analyzed. The following table shows the percentages of positive-stained follicles for the different factors studied. A total of 4,090 follicles were identified: 429 from fresh fragments, 761 from grafted fresh tissue, 333 from frozen-thawed tissue, and 2,567 from grafted frozen-thawed tissue. Follicle diameters did not differ between fresh and frozen grafted tissue (primordial follicles: 45.87 and 47.93; primary follicles: 53.89 and 54.66; secondary follicles: 121.48 and 137.73). In addition, antral follicles (fresh grafts: 29; frozen-thawed grafts: 24), corpus luteum and corpus albicans were observed. Although AMH, c-kit and KL expression appears to be affected by freezing and grafting procedures, follicles can still develop up to the antral stage.

Succesful propagation of human spermatogonial stem cells in vitro

Sadri-Ardekani,H^{1, 2}; van Daalen,S¹; Korver,CM¹;Roepers-Gajadien, HL¹; Koruji,M¹; Hovingh,SE¹; van der Veen,F¹; Mizrak,SC¹; de Rooij,DG¹; Repping,S¹; van Pelt, AMM¹ Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

² Reproductive Biotechnology Research Center, Avicenna Research Institute, Tehran, Iran

Recently developed, highly effective, cancer therapy for children allows the majority of them to survive their cancer. One of the major side effects of cancer therapy in male patients is sterility. There are currently no means to preserve reproductive potential in prepubertal boys, which contrasts with adolescents and adults, for whom cryopreservation of semen prior to chemotherapy or radiotherapy is available and widely used. Therefore, establishing a human spermatogonial stem cell (hSSC) culture system to allow successful autotransplantation for young boys diagnosed with cancer is of utmost importance. As the final number of transplantable cells will influence the success rate of this technique, we first focus on the propagation of hSSC in culture. We used testicular tissue from six men undergoing bilateral castration as part of prostate cancer treatment. Testicular cells were isolated with a two steps enzymatic digestion and overnight differential plating. Testicular cells were cultured in supplemented StemPro medium. Some formed germline stem cell (GSC) clusters were taken out of the culture and subcultured on human placental laminin coated dishes.The presence of spermatogonia in the cultures was determined by immunohistochemistry and RT-PCR for spermatogonial markers (integrin-α6, integrin-β1, PLZF). The spermatogonial stem cell transplantation assay was performed using busulphan treated nude mice, as the functional test of stem cell capability. Human cells in recipient mouse testis were detected by Fluorescent In situ hybridization (FISH) using the most common human specific repetitive DNA sequence (COT) as a probe. Germline stem cell (GSC) cluster formation was observed in the testicular cell cultures of all six men in testicular cell cultures and in subcultures GSCs. Testicular cells and subcultured GSCs could be cultured for at least 15 and 28 weeks respectively, while expression of spermatogonial cell surface markers integrin-α6 and integrin-β1 (on RNA level) and spermatogonial nuclear marker PLZF (on RNA and protein levels) was maintained.The mouse transplantation assay showed successful colonization of cultured testicular cells in 4 out of 6 patients and from the subcultured GSCs in 1 out of 2 patients, indicating the presence of functional spermatogonial stem cells. By determining the number of colonies of transplanted cultured cells from early and later passages of the same culture, we found a more than 50 fold increase of hSSC in 19 days in our testicularcell culture when cultured from day 28 to 47 (passage 2 to 5) and a more than 18,000 fold increase in number of hSSC in 64 days in our subcultured GSCs when cultured from day 77 to 141 (passage 7 to 12).

This report outlines the first successful long term culture and proliferation of hSSC in vitro. This is an important step forward to future clinical application of SSC autotransplantation in prepubertal boys diagnosed with cancer to preserve their fertility.

Morphological and ultra-structural features of cryopreserved ovine ovarian tissue: deleterious effect of PROH applying different thawing protocols

Irma C. Oskam¹, Regiane R. Santos², Babak Asadi A.¹ and Peter Fedorcsak¹.

¹ Department of Obstetrics and Gynecology, Oslo University Hospital, Oslo, Norway

Life after cancer has become a reality for many young women. However, as some cancer treatments pose a threat to reproductive function, fertility preservation methods such as cryopreservation of ovarian tissue (OT) has gained attention. Although efficient cryopreservation protocols can be designed based on the cryoprotectant toxicity and freezing curves, the thawing procedure may interfere in the success of protocols using other cryoprotectants. Sheep serve as optimal model for studies in women OT cryopreservation, with 1,2-propanediol (PrOH) as a frequently used cryoprotectant for human purposes. Aim of the present study was to evaluate the morphology and ultra-structure of preantral follicles and ovarian stroma of the PrOH protocol after applying three different thawing protocols. As a positive control, an EG standard freezing/thawing procedure was applied. Ovine OT fragments were cryopreserved according to a slow-freezing protocol using 1.5 M EG or 1.5 M PrOH. EG-frozen fragments were thawed at 37°C, and cryoprotectant removed in 3-step washes of 5 min each. PrOH thawing protocols varied with respect to temperature (37, 30 or 4 degrees). Thereafter, OT was prepared for light and transmission electron microscopic (TEM) analysis. Preantral follicles and ovarian stroma were evaluated. Data was statistically evaluated by using ANOVA and Tukey’s multiple comparison test with P < 0.05. In total, 545 preantral follicles were analysed. Cryopreservation of OT in presence of EG maintained the percentages of morphologically normal preantral follicles similar to control values (72%). Independently of the thawing temperature, OT cryopreservation in presence of PrOH did significantly reduce the percentages of normal follicles when compared to control or with those cryopreserved in presence of EG. Additionally, irreversible stroma damage was observed after OT freezing in presence of PrOH. Although the use of PrOH did reduce the percentages of normal follicles, thawing and removal of cryoprotectant at 30 degrees was significantly less deleterious than thawing at 37 or 4 degrees. All observations were confirmed by ultrastructural analysis. The use of PrOH as cryoprotectant may bring stromal and follicular deleterious effects. Although thawing at 30 degrees may minimize this effect, use of other cryoprotective agents, e.g. EG, is indicated in order to provide a safe fertility preservation method in women.

Minimal residual disease in cryopreserved ovarian cortex from patients with leukaemia

Mikkel Rosendahl, MD, Ph.D¹, Morten Tolstrup Andersen, MSc², Mette Klarskov Andersen, MD, DMSc², Lars Kjeldsen, MD, DMSc³, Elisabeth Ralfkiær, MD, DMSc⁴, and Claus Yding Andersen, MSc, DMSc¹

1) Laboratory of Reproductive Biology, 2) Department of Clinical Genetics, 3) Department of Hematology, 4) Department of Pathology. Copenhagen University Hospital—Rigshospitalet, Copenhagen, Denmark

Cryopreservation and autotransplantation of ovarian tissue in cancer patients has been justified when the patients had solid tumours and localized disease, as the risk of malignant contamination of the ovarian tissue in these cases was considered low. However in the case of leukaemia, which is a disease of the blood and hence is ubiquitous, the risk of contamination is higher and there is casuistic evidence that malignant cells are actually present in the cryopreserved ovarian cortex. The aim of this study was to systematically search for malignant cells in cryopreserved ovarian cortex from Danish women with leukaemia. Of the 37 (7 deceased) patients in our program diagnosed with leukemia (AML (Acute Myeloid Leukemia), ALL (Acute Lymphoblastic Leukemia), JMML (Juvenile Myelomonocytic Leukemia and CML (Chronic Myeloid Leukemia)) 26 (87%) agreed to participate after receiving written and oral information. Results of immunohistochemical and genetic examinations of the blood and bone marrow of the individual patients during active disease were used to guide the investigation of residual malignant cells in the cryopreserved ovarian tissue. In eight cases, a specific chromosomal abnormality could be used for detection of malignant cells by polymerase chain reaction (PCR). Of the eight patients with known chromosomal abnormalities in the malignant cells, six patients (75%) had evidence of minimal residual disease by PCR: Four CML-patients, one patient with ALL and one patient with AML. In one case of CML and one case of ALL, PCR did not reveal malignant cells. Histology and immunohistochemistry did not reveal malignant cells in neither the six PCR-positive patients, nor in the remaining 20 patients. In 75% of the cases, where a positive control was available, malignant cells were found in the cryopreserved ovarian cortex. The viability and maligant potential of these cells remains to be disclosed. For the time being however, re-implantation of tissue to leukaemia patients cannot be recommended.

From the analysis on 5571 autopsy findings of females under the age of 40 in Japan

Kyono K.¹⁾, Nakajo, Y.¹⁾, Ishikawa T¹⁾, Toya, M¹⁾, Sato Y¹, Sato K¹⁾, Akahira J.²⁾ , Sasano H.²⁾ Kyono ART Clinic¹⁾, Sendai, Japan

Department of Pathology, Tohoku University Graduate School of Medicine ²⁾, Sendai City, Japan

By July 2009, six babies had been born following ovarian cryopreservation and reimplantation in patients with Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, Ewing’s sarcoma, and sickle cell anaemia. Ovarian cryopreservation and autotransplantation could be of potential value for preservation of fertility in cancer patients; however, minimal residual disease in grafts also poses serious clinical problems in the autotransplantation of stored tissues. The scientific determination of indications is considered very important. We examined the percentages of ovarian metastasis in 5,571 autopsy files of females under the age under 40 with malignant diseases by reviewing the national autopsy files in Japan from 1981 to 2005. Total ovarian metastasis was detected in 22.4% (1,250/5,571) of all the patients with malignancies. In our data, 4.3% (5/115) of patients with Hodgkin’s lymphoma and 9.8% (5/51) of patients with non-Hodgkin’s lymphoma had ovarian metastasis. It is strongly suggested that ovarian tissue should be actively cryopreserved for fertility preservation, but we should autotransplant stored tissue with much caution until we establish reliable methods to detect minimal residual disease in grafts in a precise and reproducible manner.

Glucose/lactate metabolism as a general marker for ovarian tissue survival after cryopreservation of an intact ovary

Gerritse, R., Beerendonk, C.C.M., Braat, D.D.M., Westphal, J.R. and Peek, R.

Dept. of Obstetrics and Gynecology, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands

Transplantation of cryopreserved intact ovaries is an option for restoring fertility after sterilizing cancer therapy. Obviously, rigorous optimalisation of freeze- and thaw-protocols is a prerequisite for this approach. Thus far, studies have focused mainly on preserving the primordial/primary follicle pool, that is required for restoring fertility after auto-transplantation. However, the optimal survival of stromal tissue, that makes up more than 95% percent of the ovary, should be assured as well. We determined glucose consumption and lactate production as possible quantitative markers for the survival of ovarian tissue before and after cryopreservation.

Tissue fragments (cortex, subcortex and medulla) derived from fresh and cryopreserved bovine ovaries were cultured for 4 or 7 days. Glucose consumption and lactate production were determined in culture supernatants, on a conventional blood analyzer. Our data show that glucose and lactate levels could be readily and reproducibly measured in culture supernatants, with minimal inter-ovary variation. When freezing intact ovaries without any cryoprotectants, only low levels of glucose/lactate metabolism were observed, indicating limited tissue survival. When the ovary was submerged in medium containing DMSO prior to freezing, cortical fragments showed glucose/lactate metabolism levels identical to those observed with fresh tissue fragments. In contrast, subcortex and medulla were not protected. Preliminary experiments indicate limited protection of subcortex and medulla after perfusing the ovary with DMSO prior to freezing.

Our experiments indicate that glucose/lactate metabolism is an indicator of the amount of cryodamage sustained by the entire ovarian tissue mass. Therefore, quantitative analysis of glucose/lactate metabolism is a relatively simple and reproducible method for evaluating cryodamage. As a consequence of the high reproducibility of our assay, optimizing cryopreservation protocols for intact ovaries will require relatively few experiments.

Vitrified human ovaries harbor less primordial follicles and produce less antimullerian hormone (AMH) than slow frozen ovaries

Ozgur Oktem MD, Ebru Alper MD, Kamil Peker MD, Erhan Palaoglu MD, Basak Balaban MSc, and Bulent Urman MD

the Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Vehbi Koc Foundation, American Hospital, İstanbul, Turkey

Slow freezing is the most commonly used cryopreservation method for human ovarian tissue. Even though vitrification is being widely used in oocyte and embryo freezing, data on its applicability in human ovarian tissue freezing is limited. Therefore we aimed in this study to compare slow freezing and vitrification for human ovarian tissue freezing. 1.0 x 0.5 cm samples of healthy ovarian cortical tissue were obtained from 15 patients (mean age ± [SE] 32.2 ± 0.8) undergoing ovarian cystectomy for benign indications. Informed consent was obtained from all subjects. After dividing the tissue into 6 equal pieces two pieces were allocated into three groups (fresh, slow freezing and vitrification). A DMSO based freezing solution and a propanediol and ethylene glycol based one were used for slow freezing and vitrification, respectively. Frozen samples were thawed 24 hrs later. One piece from each group was fixed for follicle count and the other one was cultured in alpha-MEM medium with 100 mIU/mL recombinant FSH for three days. Half of the culture medium was refreshed daily and assayed for estradiol (E₂ pg/mL) by DPC and AMH (ng/mL) by ELISA. Ovarian histology, primordial follicle counts and in vitro estradiol and AMH levels were compared among fresh, slow-frozen and vitrified ovarian samples. The structure of the follicles and the stroma were preserved better in slow frozen samples compared to vitrified ones. Compared to fresh and slow frozen samples vitrified ovaries contained significantly less primordial follicles (0.97 ± 0.1 vs. 1.95 ± 0.2 p < 0.05; 0.97 ± 0.01 vs. 1.27 ± 0.1 p < 0.05; respectively); and produced significantly less AMH in vitro (0.07 ± 0.02 vs. 0.47 ± 0.2 p < 0.05; 0.07 ± 0.02 vs. 0.21 ± 0.1 p < 0.05; respectively). AMH production between fresh and slow frozen samples were comparable (0.47 ± 0.2 vs. 0.21 ± 0.1 p > 0.05). E₂ production from slow frozen and vitrified ovaries were similar (1,578 ± 270 vs. 2,120 ± 303 p > 0.05), but they were significantly lower than fresh cultured samples (1,578 ± 270 vs. 3,706 ± 943 p < 0.05 and 2,120 ± 303 vs. 3,706 ± 943 p < 0.05). Perturbed ovarian histology, more primordial follicle loss and impaired AMH production after vitrification may suggest that it may not be a suitable cryopreservation method for human ovarian tissue banking.

Which are the ideal donor and recipient vessels for a whole ovarian transplantation?

Ploteau, S.^1,2; Rogez J.M.¹ and Lengelé, B.³; (1- Laboratoire d’Anatomie, Nantes, France ;2- Department of Gynecology-Obstetrics, Nantes, France; 3- Morphologie expérimentale, Clin. Univ. saint-Luc, Brussels, Belgium)

Topic of this abstract is "Whole ovary transplantation or artificial ovary: from animal studies to human".

Our study was aimed to compare deep circumflex iliac (DCI) and deep inferior epigastric (DIE) pedicles as potential recipient vessels for a whole ovarian microvascular transplantation (WOT).

Anatomical dissections were performed on 10 fresh human female cadavers. In 3 of them, vascular injections were carried out with red or bleu colored latex or Altufix P10, in order to emphasize arteries and veins and to compare, after dissection, the morphological characteristics of the three pedicles under study. Fourteen ovaries were harvested on their gonadic (G) vascular pedicle, together with the homolateral DCI and DIE pedicles. Histological analysis was then performed on serial sections performed through all these pedicles in order to measure the diameter of the vessels at regular intervals along their whole length. Calibre values were then compared between donor (DCI or DIE) and recipient (G) pedicles, aiming to determine the optimal size match (1:1 to 1:1,3) between them. Anatomical size match is indeed one of the most prominent technical factor influencing the success rate of any microvascular procedure in tissue transplantation. Vascular injections allowed to highlight a very tortuous appearance of the gonadic artery. This arterial morphology contrasted with the venous system which always included 2 or 3 straight veins, one of them usually being wider than the others. We assessed the distance on the lombo-ovaric pedicle where the numerous gonadic vessels converged into a wider artery and vein. This critical point was located in all specimens at about 5 cm from the ovary. Individually, an optimal size match existed between gonadic and DCI arteries and veins sections among 13 out of 14 gonadic pedicles. Finally, we determined the average vascular caliber of the gonadic donor vessels and both potential recipient pedicles. On the gonadic pedicle, between 3 to 6 cm from the ovary, the artery had a constant diameter of about 1 millimeter (SD: 0,2). Mean diameter of the DCI artery was 1,25 mm (SD : 0,1), while this value raised up to 1,51 mm (SD : 0,17), for the DIE artery. Similary, the medium transverse section of the gonadic vein measured 1,20 mm, closely comparable to the DCI vein diameter which was 1,18 mm. This morphological study demonstrates that a safe microsurgical whole ovarian transplantation is feasible if the gonadic pedicle is harvested with a minimal length of 5 cm from the ovary. At this point, the pedicle contains indeed at least one artery and one dominant vein with an average section of 1 mm. Furthermore, the DCI pedicle seems to be show the best size match with the ovarian vessels in order to perform a reliable to a termino-terminal microvascular anastomosis. Additionally, rotation of the DCI pedicle in the pelvis would allow an orthotopic ovarian transplantation. Nevertheless, the DIE pedicle, although somewhat larger, may be an alternative donor site for recipient vessels if the harvest of the DCI pedicle fails or functionally delivers low blood flow vessels.

A decade of experience with fertility preservation at Karolinska University Hospital.

Rodriguez-Wallberg KA, Hovatta O.

Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden.

The fertility preservation program was created in 1998 at the Fertility Unit of Karolinska University Hospital Huddinge. Sperm cryopreservation has been carried out from 1973. Progress in assisted reproduction techniques and ongoing research protocols during these years have made possible to offer both established and experimental fertility preservation options to young patients and children facing gonadal failure. All patients who consulted for fertility preservation from 1998 to August 2009 were identified from our clinical database. Indications for fertility preservation, patient groups and interventions are described. In its early years, the program offered mainly established fertility preservation methods as sperm banking and embryo cryopreservation for adult patients. At present, hundreds of patients had benefitied from those methods. The program evolved then rapidly to offer oocyte cryopreservation to single and younger women and ovarian tissue cryopreservation to adult women and both pubertal and pre-pubertal girls. Initially, slow-freezing techniques for embryos, oocytes and ovarian tissue (Hreinsson et al. 2003) were used. In 2005 vitrification techniques started to be used systematically for cryopreservation of oocytes and in the most recent years they have been further developed to freeze ovarian and testicular tissue with promising results (Keros et al. 2005, 2007 and 2009). We have published several scientific articles from the programme. In addition, we have been developing ovarian follicle maturations methods. At present 170 female patients have frozen ovarian tissue at our unit but only three patients have returned for autotransplantation. 47 out of 67 women counseled to oocyte cryopreservation have frozen oocytes in storage. A pilot experimental testicular tissue freezing protocol, which has included 16 pre-pubertal boys is ongoing. Indications for fertility preservation have extended from malignant diseases to chronic and genetic conditions during this decade. The largest subgroup included girls with Turner’s syndrome (Borgström et al. 2009). Positive prognostic factors in this subgroup were any sign of spontaneous puberty and mosaicism. Fertility preservation options include established techniques and experimental methods. In the last ten years indications for fertility preservation have extended to chronic benign conditions and to patients who are genetically predisposed to premature gonadal failure.

Heterotopic autotransplantation of ovarian cortex in cynomolgus monkeys

Nao Suzuki,¹ Shu Hashimoto,² Suguru Igarashi,¹ Seido Takae,¹ Yoko Tsuji,² Sei Ohta, ³ Takayuki Yamochi, ³ Makoto Takenoshita,³ Yoshihiko Hosoi,⁴ Yoshiharu Morimoto,² and Bunepei Ishizuka,¹

¹ Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kanagawa, Japan, ² IVF Namba Clinic, Osaka, Japan, ³ Evebioscience, Wakayama, Japan, ⁴ Department of Genetic Engineering, Kinki University, Osaka, Japan

Orthotopic autotransplantation of ovarian cortex has advantages such as easy collection of ova and the possibility of spontaneous pregnancy. Although children have been born after successful orthotopic autotransplantation into the residual ovaries, some patients cannot undergo this procedure such as those who need bilateral ovariectomy or pelvic radiation therapy, therefore it is still necessary to investigate suitable heterotopic autotransplantation sites. The present study was performed in primates (cynomolgus monkeys) with the objective of determining the optimum site for heterotopic autotransplantation of ovarian cortex to enhance the clinical application of this method. The retroperitoneal iliac fossa and omentum were selected as sites for heterotopic autotransplantation. Two cynomolgus monkeys were subjected to laparotomy under anesthesia. After resection of the bilateral adnexae, the ovaries were cut into 0.5 cm cubes that were transplanted. Blood levels of follicle-stimulating hormone, luteinizing hormone, estradiol, and progesterone were monitored, and monkeys with a regular estrus cycle underwent superovulation and egg collection. Results: In both monkeys studied, recovery of a regular estrus cycle was confirmed after heterotopic autotransplantation of ovarian tissue. MII phase ova were successfully collected from tissues transplanted into the retroperitoneal iliac fossa or omentum. Development to the early blastocyst stage was confirmed after microfertilization. Collection of ova was still successful at a long time (935 days) after transplantation. We established an appropriate method of heterotopic autotransplantation using ovarian cortex into the retroperitoneal iliac fossa or omentum in primates. Our findings may provide useful information for the clinical application of heterotopic autotransplantation in patients without residual ovaries.

In vitro growth of isolated follicles in three dimensional alginate-collagen matrix.

Talevi R., De Stefano C.*, Barbato V., Orlando G,. Mollo V., Ferraro R. ^ф and Gualtieri R.

Dip Biologia Strutturale e Funzionale University of Naples Federico II Italy

* Centro di Fisiopatologia della Riproduzione Ospedale Moscati Avellino Italy

^ф Centro Genesis Caserta italy

Recently, many efforts have been done to help young patients to preserve their fertility through the storage of their own germ cells before chemo-and/or radiotherapy partially or completely destroys their follicular reserve. Several cryopreservation protocols have been developed for whole ovary or ovarian cortical strip cryopreservation. Unfortunately, these techniques are associated with the risk to cryopreserve also malignant cells. This risk can be avoided only if the follicles are isolated from the ovarian stroma. On the other hand, the extracellular matrix plays a key role for the development and the fully competence of follicles in vivo. The development of technologies that support the growth and maturation in vitro of oocytes from isolated follicles is attractive for both fertility preservation in oncological patients and human assisted reproduction. Herein we investigated the effectiveness of three dimensional matrix alginate and alginate + collagen to support the in vitro growth of encapsulated isolated primary and secondary follicles in bovine as animal model. Bovine cortical ovarian tissue was dissected in small pieces of 0.5 mm x 0.5 mm x 1 mm, digested with collagenase type 1A, 1 mg/mL, and DNAse, 0.2 mg/mL, at 37°C for 45 min. Single follicles were collected under the stereomicroscope and encapsulated in alginate 2% or alginate 2% + collagene IV 0,3 mg/mL. The encapsulated follicles were cultured in Medium 199 + 20% FCS in 5% CO₂ atmosphere at 38°C for ten days and the follicles growth was measured daily with computer assisted image analyzer. The viability was assessed through fluorescent labelling with propidium iodide. Morphology was evaluated under Hoffman modulation contrast and transmission electron microscopy (TEM). At the end of the culture period, the average growth and viability of follicles were 57.1% and 44% for 2% alginate and 70.3% and 71,4% for alginate 2% + collagene IV respectively. Moreover TEM analysis showed that the three dimensional follicle architecture was better preserved after alginate + collagen encapsulation. The encapsulation in a three dimensional matrix of alginate + collagen better supports the in vitro growth of isolated follicles in a bovine model. These data indicate that compounds of the extracellular matrix play a key role in the modulation of survival, growth and morphological organization of mammalian follicles in vitro and should be taken into account to improve the biotechnology for the in vitro growth of isolated human follicles.

Mixed origin of neovessels and host-graft vascular link-up in human ovarian xenografts

Van Eyck, AS.; Pham, HMC.; Dolmans, MM.

Gynaecology research unit (Prof Donnez), Université Catholique de Louvain, Brussels, Belgium

Ovarian graft behavior depends on the establishment of neovascularization. The aim of the present study was to characterize the angiogenic process leading to vascularization of human ovarian xenografts. Twenty-eight nude mice were intraperitoneally grafted with frozen-thawed human ovarian tissue obtained from 7 patients. Four time points were analyzed: days 3, 5, 9 and 21 after grafting. Host and graft vascularization were assessed by anti-mouse CD34/ anti-human CD34 double staining, followed by vascular morphometry. Double fluorescence in situ hybridation with species-specific probes, combined with immunostaining, confirmed the species origin of the cells. The endothelial cell proliferation index (EPI) and pericyte coverage index (PCI) of human vessels were assessed by human-specific CD34/Ki67 and human-specific CD34/α-SMA double staining respectively. Non-grafted ovarian tissue was included as control. The Kruskal Wallis test and χ² test were applied for statistical analysis where appropriate. P < 0.05 was considered statistically significant. Murine neovessels appeared on day 3, had significantly increased by day 5 (p < 0.05) and persisted until day 21. Human vessels persisted after grafting and a significant increased in human microvascular density was observed on day 21 (p < 0.05). From day 5, chimeric vessels composed of both murine and human endothelial cells were observed. These structures persisted until day 21. Human angiogenesis was confirmed by a significant increase in the human EPI on days 5 and 9 (12.8%–13.9%) compared to fresh tissue (1.2%, p < 0.001), returning to basal levels by day 21 (1.7%). The PCI of human vessels was shown to have significantly increased on day 9 (29.7 %) and day 21 (50.3 %) compared to fresh ovarian tissue (13.2 %, p < 0.001). Our results suggest that the mechanism of ovarian graft revascularization is a combination of inosculation (vascular link-up between graft and host vessels) and host and graft angiogenesis. Increased pericyte coverage could be a result of the ovarian graft adapting to the ischemia-reperfusion damage to promote a better blood supply.

Effects of Cholesterol-Loaded Cyclodextrin during Freezing Step of Cryopreservation with TCGY Extender Containing Bovine Serum Albumin on Quality of goat Spermatozoa

Fardin Amid*, Abbas Farshad, Abbas Koohi Khor, Sara lotfi

^*Address for corresponds: Department of Anatomy, Faculty of Medicine, Tehran University of Medical sciences,Tehran Iran.

The susceptibility of mammalian spermatozoa to cold shock and freezing damage is due to changes in membrane lipid composition, particularly cholesterol depletion in plasma membrane during cryopreservation. This study was to investigate the effects of different concentration of cholesterol-loaded cyclodextrin (CLC) and bovine serum albumin (BSA) on cryopreservation goat spermatozoa in Tris-citrate egg yolk extender. Semen was collected from four mature goats and divided into seven aliqutes prior to cryopreservation .The first aliquote remain untreated and was mixed with TCG, the second aliquote was mixed with TCG and egg yolk (TCGY), third aliquote was mixed with TCGY and 2.5% BSA (TCGYB) and other aliquotes were mixed with TCGYB containing 0.75, 1.5, 2.5 and 3 mg/ml CLC. Sperm motion Kinetics was measured by computer-assisted semen analysis (CASA) (percent motility [MOT], curvilinear velocity[VCL], straight-line velocity [VSL], average path velocity [VAP], linearity[LIN], and amplitude of lateral head displacement ALH). Acrosome status and vitality was observed by the triple-stain technique. CLC addition to extender resulted in significant (P < 0.05) enhancement of MOT, STR, and VCL of post-thawing sperm. Post-thaw Motility, progressive motility and recovery rate were significantly (P < 0.05) higher in 1.5 mg/ml CLC with 2.5 % BSA in TCGY extender compared to other groups. The 1.5 CLC sperm yielded a significant increase in percentage of spermatozoa with intact acrosome (P > 0.05). These results indicate that treating goat sperm with CLC and BSA in TCGY extender improved motility and vitality after freezing and thawing. Key words; Cholesterol-loaded cyclodextrin, Bovine serum albumin, goat spermatozoa, Cryopreservation.

Delivery rates in MS patients treated by TESE-ICSI

Carone, D.; Vizziello, GM.; Chiappetta, R; Vitti, A.

Center of Reproduction and Andrology (CREA), Taranto,Italy

The purpose of this study was to assess cumulative delivery rates in patients with non-obstructive azoospermia with multiple sclerosis in interferon treatment following treatment by testicular sperm extraction (TESE)–ICSI previous cryopreservation of male fertility with testicular biopsy.

MATERIAL AND METHODS: A cohort follow-up study was conducted. Between January 2004 and December 2008, 16 couples with non-obstructive azoospermia underwent a total of 39 fresh TESE–ICSI treatment cycles in course of interferone therapy . In addition, 20 cryo TESE–ICSI treatment cycles were performed in 13 couples for similar non-obstructive azoospermia after a period of suspension of interferon therapy . This study included only patients in whom sperm was recovered. In both groups , only patients with maturation arrest, atrophic sclerosis and germ cell aplasia were included. The main outcome measure was a delivery beyond 25 weeks gestation. In patients with fresh TESE–ICSI treatment cycles, the crude delivery rate after three cycles was 17% while the expected cumulative delivery rate was 28% [95% confidence interval (CI), 41–55]. On the other hand, in patients with cryo TESE–ICSI treatment cycles, the crude cumulative delivery rate after three treatment cycles was 27% while the expected delivery rate was 31% (95% CI, 15–46). A high dropout rate in couples with both fresh or cryo TESE-ICSI was observed (75 and 50% respectively, after the first cycle). This study shows that there is a value in performing cryo TESE–ICSI attempts in patients with obstructive azoospermia after a period of suspension of interferon therapy.

Reconstitution of spermatogenesis from testis failure after transplantation of germinal cells for male fertility preservation-a transgenic mouse model

Chen, CH ¹, Shang, WJ¹, Tsai, YC³, Wu, GJ¹, Liu, JY¹, Tzeng, CR²

¹Departments of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan ² Center for Reproductive Medicine & Sciences, Taipei Medical University and Hospital, Taipei, Taiwan, ³Center for Reproductive Medicine, Chi Mei Medical Center, Tainan, Taiwan

Fertility preservation for prepubertal male is feasible by the reconstruction of spermatogenesis after spermatogonial stem cells transplantation.

Material and Methods

1. Donor cell preparatioin

Cells for transplantation are obtained from FVB/N-Tg (PolII-luc) Ltc transgenic mouse testes of mice 5 to 60 days after birth by a two-step enzymatic digestion protocol. The cell volume needed to inject both testes of a mouse ranges from 0,1 to 0,5 ml depending on the injection method. Cell concentrations of up to about 300xl0⁶ cells per ml can be used. The cells are maintained at SCC until the time of loading into an injection pipette, usually 1 to 4 h.

1. Transplantation of SCC into recipient mouse

In this prcedure, donor cells are harvested from the testes of fertile donor mice that express a reporter transgene, and a single cell suspension of the cells is microinjected into seminiferous tubules of FVB/NJNarl wild type recipient infertile adult mice after busulfan-induced testis failure.

1. In vivo tracking of grafted SCC by bioluminescence imaging

Reporter genes can be used to assay for the activity of a particular RNA Polymerase II promoter (PolII) in each viable cell. Reporter gene products are luciferase (enzymes). The enzyme luciferase catalyzes a reaction with a luciferin to produce light which is quantified as quantum. The reporter gene is simply placed under the control of the target promoter (PolII) and the reporter gene product's activity is quantitatively measured. After gonadal tissues or germ cells transplantation, each mouse was imaged every other day for 2 weeks and subsequent every weeks for 2 months. For imaging of mice with gonadal tissue transplants. Mice were anesthetized with isoflurane. A saturating concentration of the substrate D-luciferin was injected intraperitoneally (150 mg/kg). Bioluminescence was quantified the quantum by summing pixel intensities within equal area ROI.

1. Fertilization assay

Mating the recipient male to a wild-type female, then progeny is produced by nature fertility.

Live pup of FVB/N-Tg (PolII-luc) Ltc transgenic mouse were born and imaged by bioluminescence after mating FVB/NJNarl female wild type and male wild type recipient 4–5 month after FVB/N-Tg (PolII-luc) Spermatogonial stem-cell transplantation could be used to restore fertility in men following chemotherapy or radiation treatment. Development of techniques for the in vitro differentiation of spermatogonial stem cells to functional spermatozoa is a crucial step for the treatment of infertility or germline gene therapy.

Effective cryopreservation of prepubertal mouse testicular tissue by vitrification

Curaba, M.; Camboni, A.; Amorim, C.; Wyns, C. and Van Langendonckt, A

Gynecology Research Unit (J. Donnez), Université Catholique de Louvain, Brussels, Belgium

Cryopreservation of testicular tissue from prepubertal boys with cancer has emerged as a promising approach to preserve fertility prior to gonadotoxic treatment. Our aim was to set up a vitrification protocol and compare this ice-free system with an established freezing protocol in a prepubertal murine model. Testicular tissue from 6-day-old mice was cryopreserved by slow freezing (SF, n=10) [DMSO (0.7 mol/l) + sucrose (0.1 mol/l)] or vitrification (V, n=10) [DMSO (2.8 mol/l) and EG (2.8 mol/l)]. Lactate dehydrogenase (LDH) released from damaged cells was evaluated as a measure of viability. Apoptosis (caspase-3) and proliferation (Ki67) were assessed by IHC as indicators of tissue survival, after 1 day (D1) and 3 or 10 days (D3-D10) respectively of organotypic culture. Germ cell differentiation was evaluated by LM after D10 as a marker of functionality, while tubular cell density, diameter and integrity served as parameters of structural preservation throughout culture. Ultrastructural preservation of spermatogonial, Sertoli and Leydig cells was assessed by TEM on D10. Data were expressed as medians and percentiles [P25 and P75]. The Mann-Whitney U-test was employed for statistical analysis. P ≤ 0.05 was considered significant. Before D1, specific and transitory damage caused by each treatment was identified. The percentage of released LDH revealed SF to have a more “necrotic” effect than V (54.6 [51.8, 58] vs 26.7 [23.7, 28.6]) (p ≤ 0.01), while the mean number of caspase-3-positive cells/tubule showed V to have a more “apoptotic” effect than SF (2.13 [1.17, 2.40] vs 0.07 [0.05, 0.09]) (p ≤ 0.01). On D1, both SF and V groups demonstrated a decrease in tubular cell number compared to fresh cultured controls (FR) (p ≤ 0.01), but these differences were not subsequently observed. On D3, a marked increase in proliferation was seen for both SF and V groups vs FR (p ≤ 0.01), which was not subsequently detected. Up to D10, no change in tubular diameter or integrity was noted between the SF, V and FR groups. On D10, pachytene spermatocytes were the most advanced germ cell stage observed and ultrastructure of testicular cells does not seem to be altered in FR as well as SF and V tissues. Using an original long-term organotypic culture model, we demonstrated that prepubertal mouse testicular structure and exocrine function up to the pachytene spermatocyte stage are preserved equally well by V and SF. Further investigation should be performed in vivo to assess the yield of this fast, cheap and convenient method in terms of complete germ cell maturation and sperm fertilization capacity before its implementation in human tissue cryopreservation.

Amifostine-doxorubicin association effects on prepubertal rat testes: long term damage on sperm DNA integrity and early embryo developmental delay.

Vanessa Vendramini¹, Sandra M. Miraglia¹ and Bernard Robaire².

¹ Discipline of Developmental Biology, Federal University of Sao Paulo, Brazil.

²Department of Pharmacology and Therapeutics, McGill University, Canada.

Amifostine have been applied to reduce unwanted sequelae from toxicities that can occur after chemotherapy protocols, including doxorubicin-induced cardiomyopathy. Over the past 20 years, increasingly survival rates have been reported after neoplastic malignancies occurrence during childhood. Doxorrubicin, a potent anticancer anthracycline that is widely used to combat childhood leukemia, is known to produce spermatogenic damage even in low doses. Although some studies have suggested that amifostine does not confer protection to doxorubicin-induced testicular damage, schedules including this cytoprotector have yet been applied to reduce other chemotherapy toxicities in children. Prepubertal rat testes (30-day-old) were analyzed 64 days after treatments, by accessing some microscopic morphometric parameters and sperm structure integrity (SCSA); the fertility status during adulthood (when they were 100–130-day-old). Forty-eight male prepubertal rats were equally divided into 4 groups: Doxorubicin (5 mg/kg), Amifostine (400 mg/kg), Amifostine/Doxorubicin (amifostine 15 min before doxorubicin) and Sham Control (0.9% saline solution). Adult rats showed diminution of seminiferous epithelium height compared to Control, Amifostine and Amifostine/Doxorrubicin group. However, reduction of sperm concentration, fetus implants in utero, fertility index and early embryo developmental delay were observed in both doxorubicin-treated groups, only when compared to Control and Amifostine groups. Conversely, an increase of sperm chromatin fragmentation, as accessed by SCSA, as well as blastomere fragmentation in two different moments of preimplantation embryo development were seen in both doxorubicin-treated group. The rats from Amifostine group also presented some bad effects on fertility, however, only early embryo fragmentation number was statistically increased when compared to Control group. These results suggest that although amifostine seemed to promote a reduction of doxorubicin-induced toxicity on the seminiferous epithelium of prepubertal rats, their reproductive success can be compromised, as a consequence of damaged sperm DNA and early embryo loss, after using these two drugs. Further investigation on the heritable effects produced by damaged sperm DNA on its progeny future must be a concern and should be carried out.

Human meiotic spindle alterations following slow-cool cryopreservation

Bromfield, J.J.¹; Coticchio, G.²; Sciajno, R. ²; Borini, A²; and Albertini, D.F.¹.

¹ University of Kansas Medical Centre, Kansas City, KS, USA.

² Tecnobios Procreazione, Centre for Reproductive Health, Bologna, Italy.

Cryopreservation of human oocytes permits detailed investigations into meiotic spindle dynamics. Here we evaluated the dynamic instability of spindle microtubules under cryologic conditions and their reassembly after warming to gain further insight into meiotic spindle dynamics. Meiotic spindles from slow cooled human oocytes thawed and cultured for 0, 1, 2 and 3 hrs were processed for confocal microscopy to analyse meiotic spindle organization and chromosome alignment. Oocytes were obtained from consenting IVF patients. Oocyte cryopreservation involved exposure to 1.5 mol/l propanediol and 0.3 mol/l sucrose. After thawing, cryoprotectant dilution was performed in the presence of 0.3 mol/l sucrose and decreasing concentrations of propanediol. Oocytes were fixed immediately after thawing (time 0; n=22) or following 1 (n = 35), 2 (n=19) or 3 (n=22) hrs post thaw culture and labelled for total tubulin and DNA. Single oocytes were analysed using a Zeiss LSM Pascal confocal imaging system. Spindles were classified according to shape, length, polar constriction and chromosomes position. Chilling of MII oocytes is known to depolymerise microtubules within the meiotic spindle. Immediately after thawing (time 0), no oocytes displayed a bipolar spindles with aligned chromosomes, where as at 1 hr post-thaw, 71.4% of oocytes had recovered bipolarity and chromosomal alignment. Following two and three hours of culture the incidence of bipolar spindles with aligned chromosomes was significantly reduced compared to fresh controls (31.6% and 22.7% respectively). A significant 30.7% decrease in spindle tubulin volume was seen in all occytes following freezing. In addition to a 13.8% increase in pole-pole length in spindles with mis-aligned chromosomes a positive correlation between the number of displaced chromosomes and spindle length was witnessed in thawed oocytes. This study is the first of its kind to trace the biomechanics of human oocyte meiotic spindles following cryopreservation. Directly following thawing disorganisation of the meiotic spindle is rapidly corrected. Oocytes cultured for 1 hr post-thaw displayed an ability to reform bipolar spindles that precedes chromosomal alignment along the metaphase plate. We have shown considerable alterations in the physical properties of the spindle following freezing beyond the formation of a bipolar spindle. This study has allowed us to set a baseline for human meiotic spindle dynamics that should be useful in optimizing protocols for cryopreserved human oocytes. Supported by K-INBRE, Hall family Foundation and the ESHE Fund.

Ultra-rapid vitrificaton supported follicle morphologies of cynomolgus monkeys after freezing compared to conventional vitrification and slow freezing

Hashimoto, S.¹; Suzuki, N.²; Tsuji, Y.¹; Yamanaka, M.¹; Matsumoto, H. ¹; Igarashi, S.²; Takae, S.²; Ohta, S.³; Takenoshita, M.³; Yamochi, T. ⁴; Hosoi, Y.⁴; Ishizuka, B.²; and Morimoto, Y.¹

¹ IVF Namba Clinic, Osaka, ²Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kanagawa, ³ Evebioscience, Wakayama, ⁴ Department of Genetic Engineering, Kinki University, Osaka, Japan

Cryopreservation of preantral follicles in ovarian tissues has been expected to be an effective measure for preserving young women who need to undergo cytotoxic therapy. However, cryopreservation protocol has not yet been well-established. In this study, we assessed the effects of ultra-rapid vitrification (URV), vitrification and slow freezing (SF) on morphologies of cynomolgus-ovarian tissues after freezing. Ovarian-cortical sections of 7-cynomolgus monkeys were randomly allocated to control and 3 freezing groups. Vitrification solution containing 5.64 M ethyleneglycol (EG) + 5% (w/v) PVP + 0.5 M sucrose was used for vitrification. For URV, ovarian sections loaded on Cryosupport which consist of 3 fine needles were immersed in liquid nitrogen directly. For conventional vitrification, the sections were packaged in 0.25 mL straw and the straws were immersed in LN₂. The cryoprotectants used in slow freezing were 1.5 M propanediol and 0.1 M sucrose. After warming, morphologies of follicles were analyzed using light microscopy and transmission electron microscopy. The proportion of morphologically-normal follicles vitrified ultra-rapidly (93%) was higher (P < 0.05) than those of follicles vitrified in a straw (63%) and frozen by slow freezing (59%). When the ovarian cortex was vitrified ultra-rapidly, the surface ratio of lysosomes per oocyte cytoplasm (1.3%) was lower (P < 0.05) than that in slow freezing (2.6%) and similar to that in non-frozen ovaries (1.1%). In the case of conventional vitrification, the surface ratio of lysosomes per oocyte cytoplasm (2.1%) was increased (P < 0.05) compared with non-frozen ovaries (1.1%). Results of the present study indicated that URV can support the morphology of frozen preantral follicles and oocytes compared with conventional vitrification and slow freezing.

Efficiency of oocyte cryopreservation

Borghi E., Ferraretti A.P., Magli C., Feliciani E., Lappi M., Gianaroli L.

S.I.S.Me.R., Reproductive Medicine Unit, Bologna, Italy.

Since the legislation on Assisted Reproduction Technology passed in Italy in March 2004, limiting to 3 the maximum number of eggs to be inseminated and banning embryo freezing, cryopreservation of spare oocytes entered the clinical practice. Like other procedure, oocyte cryopreservation can be affected by several factors related to patient’s characteristics or to the technique itself. Here we present an analysis of some factors affecting the efficiency of oocyte cryopreservation. Between March 2004 and April 2009, a total of 5,543 spare oocytes were cryopreserved in 831 cycles using slow freezing with 1.5 M propandiol in 0.3 M sucrose. Each oocyte was individually stored. Thawing has been performed in 735 HRT cycles. According to the law, a maximum of three eggs were inseminated. Insemination was always executed by ICSI. The results were analyzed dividing the cycles according to patient age ( ≤ 29, 30–34,35–40,> 40 yrs) and number of frozen oocytes ( ≤ 3, 4–6, 7–9, ≥ 10 ). The end points considered were: transferred cycles/thawing, clinical Pregnancy Rate (PR) per transfer, Implantation Rate (IR), Ongoing Pregnancy Rate (OPR). Cumulative results. In the 735 thawed cycles, the survival and fertilization rates were respectively 69% and 74%. The transferred cycles were 604 (82% ), resulting in 15,2% PR (92/604), 9.6% IR (115/1,199) and 9.1% OPR (67/735).

Patient age. Survival, fertilization and cleavage rates were similar in all age groups. No differences were found in the number of transferred cycles, while PR and OPR showed a continuous decline with age increasing ( PR from 24% to 10% and OPR from 15% to 5%) . This is always true in ART, but with cryopreserved oocytes the age factor started to significantly affect results at very early stages, as soon patient was gather than 30 years.

Number of frozen eggs. The survival rate was not affected by this variable, but cycles with ≤ 3 frozen eggs had significantly lower chance of transferring embryos compared to other groups ( 65% vs 87%, p < 0.001 ). In the transferred cycles, PR and IR were not influenced by the number of frozen eggs, while OPR was significantly higher in patients with more than 10 fozen eggs than the other categories (14% vs 7.5%, p < 0.025). At the present time, oocyte cryopreservation is beneficial only in optimal conditions: young patients, high number and high quality oocytes. Given the efficacy of this technique, we can consider oocyte cryopreservation effective for preserving fertility in young women.

Clinical grade vitrification of human ovarian tissue

Sheikhi M, Lundqvist M, Hultenby K, Pettersson K, Hovatta O

Ovarian tissue cryopreservation helps young women who face premature ovarian failure, due to chemotherapy, radiotherapy or genetic causes. Orthotopic transplantation of human frozen-thawed ovarian tissue has already resulted in birth of several infants. Maturation of ovarian follicles in vitro is also progressing. To optimise the cryopreservation of ovarian tissue we carried out a study comparing slow rate freezing and vitrification (Keros et al. 2009). Now we have developed a closed system for a clinically safe vitrification of human ovarian tissue.

The aim of the study was to evaluate the possibility to perform vitrification according to good manufacturing practice quality system, GMP. Ovarian tissues were donated by women, aged 28–43 years, undergoing Caesarean sections. The cryoprotectants used for vitrification were a combination of dimethyl sulphoxide (DMSO), PrOH, EG and polyvinylpyrrolidone (PVP) established recently in our laboratory (Keros et al. 2009). Nunc Cryo Tube Vials with silicon ring were used as device for vitrification. The morphology of the pre- antral follicles, granulosa cells and ovarian stroma after vitrification was analysed by light microscopy in semi-thin sections.

The morphology of the vitrified tissue in cryotubes did not differ from that obtained earlier using a system in which the tissue pieces were in contact with liquid nitrogen. We observed a good preservation with intact morphology of the stroma and the neighboring stromal cells. A nucleus with no shrinkage of nuclear membranes was found. Granulosa cells had contact with a uniform basement membrane, neighboring granulose cells and oocytes. Vitrification is an advanced alternative for cryopreservation of ovarian tissue. Based on the results from this study the procedure can be carried out according to good manufacturing practice quality system. No direct contact with liquid nitrogen is necessary to achieve good preservation of the vitrified tissues. The tissues can be safely stored for the future, in closed cryotubes immersed in liquid nitrogen.

Transmission electron microscopy will be done and the results will be presented.

Effects of Ionizing Radiation on Ovulation Rate and Oocyte Morphology in Mouse

SAPMAZ METIN M., KANTER M.

Department of Histology and Embryology, Faculty of Medicine, Trakya University, Edirne, Turkey.

This study aimed to investigate the in vivo effects of ionizing radiation on maturation ability and radiosensitivity of oocytes enclosed in preantral and antral follicles, as a result of spontaneous or exogenously stimulated ovulation. Experiments were performed in 6–8 weeks age, Balb/c female mice. Mice received single dose, total body 7.2 Gy gamma radiation at the diestrous-proestrous transitition period. Irradiated animals were sacrificed in two subsequent estrous stages following irradiation, corresponding to spontaneous ovulation time. To analyse the ovulation function of the exposured ovaries, irradiated animals were superovulated in the first and second proestrous stages. Ovulated oocytes were collected and ovulation rates were obtained both of spontaneous or exogenously stimulated ovulations. Spontaneous ovulation rate of the exposed antral follicles was significantly lower than sham-irradiated mice in the first estrous stage following irradiation (p < 0.01). Furthermore, most of the oocytes were M I stage. Oocyte morphology and ovulation rate of the exposed preantral follicles were similar with sham-irradiated group in the second estrous stage following irradiation. Morphological abnormalities were observed in the oocytes and polar body as well, both in the first and second estrous, after irradiation. Superovulation response of the all irradiated animals were lower than respective control animals. Ovarian response was significantly low particularly in the first ovulation time after irradiation (p < 0.01). On the other hand, 8 of 16 irradiated animals had longer cycle than pre-irradiation (p < 0.05).

In these in vivo results indicate early signs of ionizing radiation-induced premature ovarian failure in mice. Also, in this study, estrous stages dependent applications may contribute to a better understanding of acute effects of ionizing radiation.

Expression of genes heat shock protein 70 and MnSOD during Vitrification of Mouse MII oocyte following Cryotop method

Fardin Amidi^*, Zahra khodabandeh^, Aligoli sobhani, Kobra mehrannia, Seid Mohammad Hossein Nori, Mehdi abbasi, Mahyar Habibi rodsari,

^*Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences, Tehran Iran.

The aim of this study was to investigate the effect of two vitrification protocols on mouse MII oocytes and evaluate its effects on expression of genes heat shock protein 70 and MnSOD.

The oocytes were collected and vitrified with 10 %( v/v) EG + 10 %( v/v) DMSO + 0.5 M sucrose in group A(VSI) and 14.5% (v/v) EG + 14.5% PROH +0.5 M sucrose in group B(VSII), respectively. For thawing vitrified oocytes were put into 1 M sucrose for 1 min and two diluted solution for 3 min. After thawing the oocytes were fertilized and cultured in vitro to develop in two cells. Survival rate and two cell embryos were evaluated and gene expression (HSP 70, MnSOD and β actin) was examined by reverse transcription polymerase chain reaction (RT_PCR). Survival rate of mouse oocytes after warming was lower in two groups (VSI: 91.2% ± 1.7, VSII: 89.2% ± 1.5) compared to control (100.0% ± 0.01). The rate of IVF were significantly (p ≤ 0.05) reduced in vitrified-warmed (VSI: 39% ± 5.8; VSII: 34% ± 5.7) oocytes compared to control (88.36% ± 2.3). We analyzed the expression of specific gene such as Hsp70, and MnSOD. The abundance of mRNA was generally reduced in oocytes related to vitrification procedures, but expression of MnSOD increased in vitrified-warmed compared to control oocytes. We also detected Hsp70 only in control and VSI group. Our results demonstrated that increasing HSP 70 activity in VSI might improve the developmental competence of vitrified oocytes.

Slow cooling protocol: results in oocytes cryopreservation

Bartolotti, T.¹; Camerani, M.F.¹; Felletti, V.; Di Nenno, L.¹; Cinti, M.L.¹; Schiuma, N.¹; Rambelli, V.¹

^1. AUSL of Ravenna, Lugo Hospital, viale Dante, 10 48022 Lugo, Ravenna, Italy

The study evaluated success rates derived from the use of cryopreserved oocytes by using a slow freezing / rapid thawing protocol. Since January 2007, a total of 126 oocyte freezing cycles were performed. No restriction in the selection of the patients. According to morphologic features, good quality oocytes were frozen using slow cooling protocol which make use of PrOH (1.5 M) and sucrose (0.2 M) as cryoprotectants. Oocytes cryopreservation was carried out between 1 and 2 h from oocytes retrieval. Oocytes were thawed by a rapid thawing protocol, and cryoprotectants were removed by step-wise dilutions. After 150–180 min, survived oocytes were microinjected. 52 thawing cycles were performed and a total of 289 oocytes were thawed.

The survival rate was 63%. Fertilization and cleavage rates were 66.2% and 91.3% respectively. A total of 91 embryos were transferred in 44 embryo transfers. 11 pregnancies were achieved and the pregnancy rate per transfer was 25%. 14 gestational sacs were observed at ultrasound examination and the implantation rate was 15.4%. 3 patients aborted with a rate of 27.3%. 6 babies were born and 3 pregnancies, 2 singleton and 1 twin, were still ongoing. Good results in IVF cycles performed using frozen / thawed oocytes, allow to consider oocytes cryopresevation in addition to conventional IVF. Oocyte cryopreservation represents a strategy to improve chances of success per patients, without recurring to ovarian stimulation; furthermore it rapresents an efficient methodology used to preserve female fertility for patients at risk of loosing ovarian function.

Oocyte retrieval at the time of laparotomy for oophorectomy

A Carby¹, V Hird², A Farthing², S Lavery¹

IVF Hammermsith, London UK¹, Hammersmith Hospital, London, UK².

We present two cases of egg retrieval at the time of laparotomy after controlled ovarian stimulation with subsequent fertilisation of oocytes. Both women had a diagnosis of carcinoma, one a borderline tumour of the ovary with previous unilateral oophorectomy, the other metastatic endometrial cancer requiring bilateral oophorectomy post hysterectomy. In both cases transvaginal oocyte retrieval risked pelvic spread of disease and was contraindicated. Although collection of immature oocytes and subsequent in vitro maturation after oophorectomy has previously been described this is the first report of mature oocyte collection prior to oophorectomy at the time of laparotomy. An antagonist treatment cycle was utilised for both patients. FSH stimulation (Gonal-F Serono ™) was commenced on day 2 of the menstrual cycle and LHRH antagonist (Orgalutran Organon ™) on day 5 of FSH stimulation as per protocol. FSH and LHRH antagonist were continued until the day of hCG injection (Ovitrelle Serono™). Laparoscopic oocyte retrieval was timed for 36 h post hCG injection. The abdomen was opened by a Pfannenstiel incision and the ovarie(s) isolated. Before occluding their blood supply oocyte aspiration was performed using a double channel needle (Casmed 15G™) with direct puncture of the follicles and flushing with warmed Hartmann’s solution if required. The ovaries were examined post oophorectomy and aspirated to ensure all follicles had been punctured. An ICSI procedure was then undertaken to achieve fertilisation. On the day of hCG instruction patient DT had one follicle >17 mm, patient TW 7 oocytes >17 mm. Two oocytes were retrieved from DT of which one fertilised and a single two cell embryo was transferred two days after oocyte retrieval. Subsequently serum hCG fourteen days post oocyte retrieval was negative Ten oocytes were retrieved from patient TW of which 6 fertilised including two oocytes which were retrieved post oophorectomy. All embryos were frozen at the PN stage for later use in a surrogate. Controlled ovarian stimulation and subsequent oocyte retrieval at the time of laparotomy provides a novel method of fertility preservation in those women faced with oophorectomy and for whom transvaginal oocyte retrieval is contraindicated. Oocyte retrieval and fertilisation rates appear comparable with transvaginal oocyte retrieval.

Ovarian Protection by GnRH Agonist from Gonadotoxic Chemotherapy- A Mouse Animal

Model

Chen, CH ¹ , Tang SJ WJ¹, Tzeng, CR²

¹Departments of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan ² Center for Reproductive Medicine & Sciences, Taipei Medical University and Hospital, Taipei, Taiwan

24 female sex mature FVB mice (8 weeks old) were used for this study and divided equally into four groups. The control group was prepared without administration of drugs. The Busulfan group underwent injection of Busulfan at dose of 50 mg/kg and was sacrificed four weeks later for histological examination of ovary. GnRH agonist (Triptorelin) was injected subcutaneously in the Triptorelin (low dose, 3.8 mg/kg) + Busulfan group and the 10Triptorelin (high dose, 38 mg/kg) + Busulfan group, followed by injection of Busulfan four weeks later. They were also sacrificed another four weeks later as the Busulfan group. First we compared the total number of follicles and numbers of primordial, primary, secondary, and antral follicles between the Busulfan group and the control group to see the impact of chemotherapy on ovaries. Secondly, the protective efficacy of different dosages of GnRH agonist was evaluated by comparison of the numbers of follicles the Busulfan + Triptorelin groups with the Busulfan group. The excised ovaries were fixed in formalin and then embedded in paraffin. Six-µm serial sections were taken with a microtome and stained by hematoxylin and eosin. The numbers of primordial, primary, secondary and antral follicles containing an oocyte were counted by an experienced examiner blind to condition of the animal. Data were presented as mean ± SEM and median. The significance was determined by the Tukey Method. The 95% confidence interval not including 0 meant the difference between those 2 groups was statistically significant. All the statistics was processed by STATA 8.0. Significant depletion of primordial, primary and secondary follicles was found in the Busulfan group compared with the control group with a statistical difference. We also observed obvious destruction of the ovarian tissue under the microscope. Both have demonstrated the gonadotoxicity of Busulfan. To examine if GnRH agonist could provide any protective effect on the ovaries, we used different dose of single Triptorelin injection four weeks before administration of Busulfan. In the Triptorelin + Busulfan group, larger primordial follicle count was found compared with Busulfan group without significant difference, and so was primary follicle count. In the 10Triptorelin + Busulfan group, the numbers of primordial follicles and primary follicles were significantly higher compared with the Busulfan group. Although secondary follicle count increased, the difference was not significant. Because of the pool of primordial follicles and prumary follicles making up more than 90% of follicle population, ovarian reserve preservation has been demonstrated when Triptorelin was added. The reason why the data of the Triptorelin + Busulfan group was not statistically significant might attribute to insufficient dose of Triptorelin. A larger number of primordial follicles and primordial follicles were preserved in the 10Triptorelin + Busulfan group than in the Triptorelin + Busulfan group. If there is any dose scale for protective effect of GnRH agonist on the ovary need to be tested in the future. Our animal study indicates GnRH agonist as an ovarian protection method against gonadotoxicity by chemotherapy. It also provides the first quantitative histological evidence suggesting a dose-dependent protective effect on the ovarian reserve. The number of primordial follicles is still the most direct marker for evaluating fertility and the effect of protection, but developing a more applicable surrogate is urgent. Based on the results of this study and the recent published literature, GnRH agonist co-treatment is not only a non-invasive strategy but a rational option for fertility preservation. It should be considered in all patients during their reproductive age before receiving chemotherapy.

Title: Xenotransplantation of human ovarian tissue to nude mice: comparison between four grafting sites.

Dath C, Romeu L, Delle Vigne L, Van Eyck, AS

Gynecology Research Unit—Université Catholique de Louvain—Brussels—Belgium

Transplantation of human ovarian tissue to nude mice is a useful experimental model to assess ovarian function and developmental potential of ovarian follicles after grafting. However, there is currently no consensus in the literature as to the optimal grafting site. This study was designed to assess the impact of different ovarian tissue transplantation sites on the follicular pool and ovarian tissue integrity after short-term grafting. Frozen-thawed ovarian tissue from eight patients was grafted for one or three weeks to the peritoneum, inside the ovarian bursa, under the skin, and into the muscle of sixteen nude mice. Assessment of follicular density and follicle classification were carried out by histological analysis. Proliferative activity was evidenced by immunostaining with anti-Ki-67 antibodies, and fibrotic areas were analyzed by morphometry on histological slides. Statistical analyses were performed using the Kruskal-Wallis test, Mann-Whitney test and chi-square test where appropriate. No significant differences in follicular density were evidenced between sites or days post-grafting. One week post-transplantation, the proportion of Ki-67-positive primordial follicles was higher (20–42%) than in controls (1.7%) (p < 0.05), demonstrating follicular activation in all four sites. Three weeks post-grafting, activation was lower and most primordial follicles (34.1 to 66.9% of the follicle population, depending on the grafting site) were quiescent. Cryopreservation and grafting resulted in extensive fibrosis in the stroma. This fibrosis was significantly less pronounced in intramuscular grafts, representing 18.8% of the surface, versus 44.7–60.5% for other sites, after three weeks’grafting. All four grafting sites equally supported early follicular growth and maintained a pool of quiescent follicles after short-term frozen-thawed human ovarian tissue transplantation. The extensive fibrosis observed does not appear to have a major impact on early follicle development, but its long-term effects must be investigated. The graft environment may be implicated in the preservation of the stroma, as suggested by a lower degree of fibrosis in the intramuscular site. Xenografting, ovarian tissue, follicular activation, transplantation site, stromal fibrosis.

Suppression of follicular development between ovarian grafts after auto-transplantation at different sites in humans.

Dittrich R, Hoffmann I, Oppelt PG, Mueller A, Beckmann MW

OB/GYN, Erlangen University Hospital, D-91054 Erlangen, Germany

Cancer treatment in young women has greatly enhanced their life expectancy, but these treatments often cause infertility due to a massive destruction of the ovarian reserve resulting in premature ovarian failure. One possibility to overcome sterility is to cryopreserve ovarian tissue before oncological treatment and later to transplant the tissue back into the patient. However, many unanswered questions remain regarding the development of follicles in the transplanted tissue as well as what will happen with the tissue when the hypergonadotropic condition is attained after the follicular pool is exhausted. In this study we histologically examined ovarian grafts at different transplantation sites after LH and FSH serum levels increased again. Cryopreserved ovarian tissue was retransplanted orthotopically by laparoscopy in different peritoneal pockets after a period of cancer remission. One site was near the fallopian tube, the other in the pelvic wall. 11 months after transplantation, when several cycles had passed the grafted tissue was removed because LH and FSH serum levels increased to menopausal levels. The grafts were fixed in formalin. After routine paraffin embedding, the entire samples were serially sectioned and the number of follicles as well as the presence of atypical cell formation were examined. At one site of transplantation no follicles were found, however in the other site (near the fallopian tube) primordial as well as antral follicles were found. No atypical mitosis were found in any of the grafts. There was an abundance of blood vessels and a normal stromal tissue. It is known that the transplantation of small grafts can only restore fertility for a short period of time. In this case it seems to be evident that the grafts at one transplantation site suppress the growth of follicles in the other transplantation site, since follicle growth started only after the reserve of primordial follicles was exhausted at the other site. The factors responsible for this growth inhibition are unknown and should be further investigated.

In-Vitro Growth of Large versus Small Preantral Follicles and Oocyte Maturation from Mouse Ovaries

Budi Wiweko¹, Muharam Natadisastra¹, Akiko Hasegawa²

¹Department of Obstetrics and Gynecology, Faculty of Medicine University of Indonesia, Jakarta, Indonesia

²Laboratory of Developmental Biology and Reproduction,

Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Japan

The ability to develop mature oocytes yielded from immature preantral ovarian follicles is a key success in female gamete preservation. The purpose of this study was to describe the in-vitro growth (IVG) of preantral follicles and in-vitro maturation (IVM) of oocytes taken from mouse ovarian tissue. The preantral follicles were mechanically isolated from fresh ovaries of 7-weeks-old specific-pathogen-free, female (C57/BL/6xDBA/2) F1 mice (Japan SLC Inc. Shizuoka, Japan). Those preantral follicles were cultured on IVG medium for 8–11 days then transferred to IVM medium for 16–18 h. The maturation stage of oocytes was evaluated after mechanical denudation. The preantral follicles in this study were grouped into large preantral follicles and small preantral follicles based on the growth and the expansion of granulosa cells. The antral follicles formation was found 85% in large follicles group and 33% in small follicles group. The growth of antral follicles was more likely to happen in the large preantral follicles group. This finding may be explained by the expansion of granulosa cells on day 2 IVG in large follicles which increased the probability of large follicles to develop into antral follicles. All antral follicles were matured into metaphase II (MII) oocytes (22%) and metaphase I (MI) oocytes (78%). There was no germinal vesicle oocyte found at the end of maturation process. This point showed potency of antral follicles as the yielded source of MII and MI oocytes. Mature oocytes could be yielded from preantral mouse ovarian follicles. Large pre-antral follicles at the earlier day of IVG were more likely to become antral follicle compared to small preantral follicles.

The Optimal Timing of Cryoprotectant Agent Exposure Based on Morphological Alteration In Primary Follicles from Vitrified Sheep Ovarian Cortex

Budi Wiweko¹, Iwan Kurnia¹, Andon Hestiantoro¹, Arief Boediono²

¹Department of Obstetrics and Gynecology, Faculty of Medicine University of Indonesia, Jakarta, Indonesia

²Faculty of Veterinary Medicine Bogor Agricultural University, West Java, Indonesia

Vitrification of ovarian tissue has been known as the best method in female gamete cryopreservation. The optimal technique and timing of cryoprotectant agent (CPA) exposure in vitrification should result in no alteration or minimal alteration in order to ensure the viability of gametes after cryopreservation. This study is purposed to find the optimum duration of sheep ovarian cortex exposure to CPA based on the morphological alteration in primary follicles after vitrification. The ovaries from five months old sheep were collected at the slaughterhouse and transported to the laboratory within 2 h of collection. The ovarian cortex fragments were divided into control group (no CPA exposure), 15 min CPA exposure, 30 min CPA exposure, 45 min CPA exposure and 60 min CPA exposure. The morphological alteration including disconnection among granulosa cells of primary follicles, oocytes cytoplasm, zona pellucida, and oocytes shape were analyzed. The percentage of normal follicles in those groups after vitrification were 0% in no CPA group, 58% in 15 min CPA, 95% in 30 min CPA, 1% in 45 min CPA, and 0% in 60 min CPA. The percentage of intact oocytes were 0% in no CPA group, 75% in 15 min CPA, 100% in 30 min CPA, 21.5% in 45 min CPA, and 12% in 60 min CPA. It indicated that the various timing with CPA exposure results in various outcome so that determining the best optimum timing of CPA was really important in vitrification. From that result, the best timing of CPA exposure before vitrification was 30 min. The optimal timing of CPA exposure is influential in vitrification process. From this study 30 min CPA exposure gave the best result.

Fertility preservation in a young woman undergoing chemotherapy: a new approach

Szlit Feldman E.; Ahumada A.; Arenas G.; Fava F.; Leocata Nieto F.; Asch R.

Procrearte, Buenos Aires, Argentina

Chemotherapy in young patients with breast cancer usually causes premature ovarian failure and infertility. Fertility preservation in such cases is one of the main challenges for specialists in human reproduction. Oocyte vitrification have been proposed as a technique to preserve fertility in young women .Unfortunately, since breast cancer proliferation and dissemination can be triggered by estrogen, conventional ovarian stimulation regimens are considered by many oncologists to be contraindicated in these patients. We report here the possibility of a successful oocyte cryopreservation benefiting of the protocols usually prescribed by the oncologist for the treatment of breast carcinoma. A 31 year old patient (G0P0) with an intraductal breast carcinoma T1 N0 Grade 3 Stage I, positive for estrogen receptors (80 %)(p ER), positive for progesterone receptors (20%) and positive for HER-2/neu underwent left superior quadrantectomy and sentinel nodule resection. Following surgery, and two weeks before starting with chemotherapy, in order to try to preserve her menstrual function by ovarian suppression she began the administration of Goserelin depot. Utilizing the “flare up” effect of the GnRh analogue she was monitored daily by transvaginal ultrasound (US) and at the third day, when the follicles reached 10 mm diameter, 5,000 I.U of hCG were injected and 35 h later US guided transvaginal oocyte retrieval was carried out: 6 immature oocytes were obtained. Three of them matured in vitro after 48 h and vitrified. The adjuvant chemotherapy was: 4 cycles of Adriamycin 60 mg./m2 with Cyclophosphamide 600 mg./m2 every 3 weeks followed by Paclitaxel 80 mg/m2 weekly with concomitant administration of Trastuzumab, which was continued for one year. At the end of the administration of Paclitaxel and Goserelin the patient initiated Tamoxifen 20 mg/day for five months. Following the discontinuation of Tamoxifen the patient was monitored by US and two follicles of 15 mm were observed; after 35 h of hCG 5,000 UI administration two metaphase II oocytes were harvested and vitrified. A young woman with breast carcinoma ( pER) who desired to preserve her fertility underwent two ovarian retrievals in order to store oocytes benefiting by the use of two drugs included in the oncological protocols. As a result, five mature oocytes have been cryopreserved for a potential future use. For many investigators the use GnRh analogues in parallel with chemotherapy in cancer patients has demonstrated a significant decrease in the rate of premature ovarian failure than in controls .In addition, in those patients with breast cancer the use of GnRh analogues was associated with less recurrence and improved survival rate. Tamoxifen is broadly used in premenopausal pER breast cancer patients. The possibilities to preserve fertility in women exposed to chemotherapy are limited, because none of the methods is ideal. Combination of several methods such as proposed in this report should be considered.

Assessment of ovarian reserve after radio-chemotherapy treatments in cancer patients who underwent fertility preservation

Hasson J, Amit A, Eshel A, Cohen T, Alon A, Ben-Yosef D, Lessing JB, Azem F

IVF unit, Tel Aviv Souraski Medical Center, Tel Aviv, Israel

Chemo-radiotherapy treatments are potentially gonadotoxic, thus necessitating fertility preservation. This study was aimed to assess ovarian reserve after chemo-radiotherapy in cancer patients who underwent fertility preservation, in order to establish a "tailored" approach for preserving fertility.

A retrospective study including 63 cancer patients who had undergone fertility preservation procedures. Indications for treatment included: breast cancer (13 patients, 21%), hematological malignancies (27 patients, 43%), sarcoma (9 patients, 14%), colon cancer (5 patients, 8%), cervical cancer (5 patients, 8%) and miscellaneous cancers (4 patients, 6%). Mean age was 29±1 years. 71% had no children. 48% underwent OTCP (ovarian tissue cryopreservation), 62% were treated with GnRH analogs, 30% underwent IVF with embryos cryopreservation (of them 20% underwent also oocytes cryopreservation) and 10% underwent ovarian transposition. In 47% of patients a combination of several techniques was applied. 1 year after chemo-radiotherapy breast cancer patients mean FSH level was 9.6 ± 7 mIU/L; mean antral follicles count (AFC) 2.7±2 and mean Anti Mullerian Hormone (AMH) level 5.2±4 ng/ml.12 pregnancies and 10 live births were achieved. Hematological malignancies patients mean FSH level was 36±24 mIU/L, mean AFC 5 ± 3 and mean AMH 9 ± 6 ng/dl.8 pregnancies and 7 live births were achieved. Sarcoma patients mean FSH level was 14 ± 7 mIU/L, mean AFC 1.8±1 and mean AMH 6 ± 4 ng/dl. 3 pregnancies and 1 live birth were achieved. Colon cancer patients had normal ovarian reserve while cervical cancer patients showed premature ovarian failure parameters. Following chemotherapy, breast cancer patients have preserved ovarian reserve; thus, procedures like OTCP seem to be unjustified, while sarcoma patients have low post treatment ovarian reserve, hence OTCP and other procedures are justified. Hematological malignancies patients show heterogeneous results with diminished ovarian reserve parameters but good recovery as expressed by a relatively high rate of pregnancies and live births. In this sub-group of patients OTCP seems to be unjustified and GnRH analog treatment should be considered.

Ovarian tissue cryopreservation (OTCP) for fertility preservation in cancer patients-is it justified in advanced stages?

Hasson J, Ben-Yosef D, Cohen T, Eshel A, Lessing JB, Amit A, Azem F

IVF unit, Tel Aviv Souraski Medical Center, Tel Aviv, Israel

Ovarian tissue cryopreservation (OTCP) has been developed to sustain reproductive function of women who face sterilizing chemotherapy, radiotherapy or radical surgery. This study was aimed to asses the impact of advanced cancer stage among a cohort of cancer patients who underwent OTCP for fertility preservation. Retrospective study including 54 cancer patients who underwent OTCP for fertility preservation in our institution. Indications for treatment were: breast cancer (n=16), sarcoma (n=14), Hodgkin's disease (n=10), cervical cancer (n = 3), colon cancer (n=2), miscellaneous cancers (n = 9). Co-treatment with monthly injections of GnRH agonist during chemotherapy was also administered. Mean age of patients was 26 ± 6 years. Based on history, physical examination, imaging and diagnostic work up, 68% of patients were in early stages of disease (up to stage II) when OTCP was performed. Twelve patients (21%) passed away during follow up: six patients died from osteosarcoma (6/14, 43%), 3 of them where staged as local disease (up to stage II) at diagnosis. Two patients died from breast cancer (2/16, 12.5%), both had metastasis at diagnosis. One patient died from vulvar cancer (1/1, 100%), found to be stage IV at surgery at which OTCP was also performed. One patient died from acute leukemia (1/1, 100%) and one patient died from cervical cancer (1/3, 33%) which was found to involve lymph nodes at surgery. One patient died from colon cancer (1/2, 50%), initially diagnosed as stage I but found to be stage III at surgery. All together, 4 patients of the 12 patients who died (25%) were initially diagnosed as having local disease at the time OTCP was performed. Thus far, studies regarding OTCP focused on the negative impact of radio/chemotherapy on future sterility, without taking into account the expected prognosis of patients. The fact that 75% of patients who died were at advanced stages of disease when diagnosed, and that the other 25% had local disease but still died raises two major issues: First, the limitations of pretreatment evaluation and secondly an ethical question regarding proper selection of candidate patients for OTCP. Indeed, if fertility is to be preserved, one should first be as sure as possible that life is preserved in the first square. We believe, thus, that the expected prognosis of the basic cancer disease is an important component in the decision whether to perform OTCP and that patients with unfavorable prognosis are not candidates for this procedure. It seems that when prognosis is poor, minimal intervention to preserve fertility may be more appropriate, and OTCP may be unjustified.

Melatonin promotes the cumulus–oocyte complexes quality of vitrified–thawed murine ovaries; with increased mean number of follicles survival and ovary size following heterotopic transplantation.

Masoud Hemadi a, Farid Abolhassani, Mohammad Akbari, Aligholi Sobhani, Parichehr Pasbakhsh, Lars Ährlund-Richter, Mohammad H. Modaresi , Mojdeh Salehnia d

Department of Anatomy, Faculty of Medicine, Medical Sciences/University of Tehran, Tehran, Iran.

We have tested the protective effect of melatonin on neonate murine ovarian tissue after vitrification, thawing and heterotopic transplantation into ovariectomized recipient mice.

Vitrified ovaries from neonate (CBA × C57Bl/6) F1 hybrid mice were thawed under standard condition with or without the addition of 100 µM melatonin. Following transplantation, melatonin (20 mg/kg/day) or saline solution (physiological saline) was injected i.p. to the treated and non-treated groups for 48 h respectively. Follicle survival and development, together with ovary size followed. Also, vaginal cytology was carried out for monitoring restored puberty.

Results: Histological and immunohistochemical studies showed that melatonin could promote the quality of the cumulus–oocyte complexes with uniform distribution of granulosa and stromal cells in the ovarian grafts. Furthermore, the mean follicles survival was improved and the ovary size increased (P≤0.001). The overall mean number of follicles entering the next maturation stage dramatically increased. However, the revascularization and restoration of puberty of ovarian grafts were similar between melatonin- treated and control groups.

: melatonin as a protection from ischemic injury and a reduce oxidative stress, was shown beneficial during the early days of transplantation.

Effect of antifreeze protein on ovarian tissue cryopreservation: A preliminary study

Jee, B.C.^{1, 2}; Lee, J. R.¹; Youm, H.¹; Suh, C.S.^{1, 2}; Kim, S.H.²; Moon, S.Y.²

¹Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam, Korea

² Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, Korea

Antifreeze proteins (AFPs) are a class of polypeptides produced by certain animals such as antarctic fish which permit their survival in subzero environments. Recently, several studies on protective effect of AFP for cryopreservation of animal cells and organs were reported. However there has been no study on antifreeze protein for ovarian cryopreservation. The purpose of this study was to investigate the effect of AFP supplementation during ovarian vitrification procedure. Ovaries from 4 week-aged ICR mice were vitrified using a two-step exposure to equilibrium and vitrification solutions. Equilibrium solution was 20% ethylene glycol (EG) and vitrification solution was composed of 40% EG, 18% Ficoll and 0.3 M sucrose. All solutions were based on Dulbecco’s phosphate buffered saline containing 20% fetal bovine serum. Ovaries were divided into two groups (AFP and control) according to whether AFP III (5 mg/mL) was supplemented to the solutions. After exposure to solutions, ovaries were transferred to cryovials and plunged into liquid nitrogen. Two weeks later, the ovaries were thawed and follicular morphology and apoptosis were analyzed and compared to control group. Morphology was graded into three (grade I, II, III) and apoptosis was assessed using TUNEL assay. A total of 487 and 588 follicles from 22 ovaries (10 from control 12 from AFP group) were analyzed for morphologic and TUNEL assay, respectively. The rate of follicle with grade I morphology was significantly higher in AFP group (41.4% (99/239) vs. 28.6% (71/248), p = 0.003). Apoptotic follicle (TUNEL-positive) rate was significantly lower in AFP group compared to control group (19.3% (59/305) vs. 27.6% (78/283), p = 0.019). Our data suggest that AFP supplementation could improve tissue survival after ovarian vitrification.

Managment of fertility-preservation in breast-cancer-patients under 40 years in a large breast cancer center

Lawrenz B.; Neunhoeffer E.; Henes M.; Lessmann-Bechle S.; Fehm T., University womens hospital Tuebingen, Germany

The increase of breast cancer in young women under 40 years and the increasing age of women at the time of the birth of their first child underlines the importance to implement counseling for fertility-preserving strategies in the management of breast cancer care. We present the fertility preserving procedures performed after routine counseling for primary breast cancer patients in a large breast cancer center (approx. 600 primary cases per year). Between November 2006 and June 2009 42 patients under 40 years with histologically confirmed breast cancer were counselled for the fertility-preserving possibilities before breast surgery and chemotherapy in the fertility-center of the university womens hospital in Tuebingen. In 32 months 42 primary breast cancer patients were counselled. The majority of the patients (n = 17) decided for ovarian tissue cryopreservation as fertility preserving method. The laparoscopic approach was normally combined with the oncological—surgical procedure. GnRH-protection was performed in 10 patients. In 9 patients an ovarian stimulation protocol was initiated to cryopreservate fertilized or unfertilized oocytes. A combination of different fertility-preserving methods was performed in 9 patients. The majority of the young primary breast cancer patients in our study were hormone receptor positive. Because of this special tumor entity the possibilities to perform fertility-preserving methods is very limited especially in those patients, who have to undergo neoadjuvant chemotherapy. The only therapeutic option for fertility preserving methods in this patient group is the ovarian tissue cryopreservation.

Patient experiences with fertility preservation in cancer: satisfaction in general but unmet information needs.

L.A. Louwé¹, M.M. ter Kuile¹, E. Jenninga¹, C.G.J.M. Hilders², A.M. Stiggelbout³

Interest in fertility preserving therapy (FPT) in female cancer patients has grown since the birth of a healthy baby girl after transplantation of cryopreserved ovarian tissue. Limited knowledge is available on the decision-making process regarding fertility preservation therapy. The aim was to examine perceptions of the decision-making process of young female cancer patients who were informed about female fertility preserving options, and to assess patients’ information needs.

We conducted retrospective semi-structured interviews with women with cancer (18+) who had had at least one face-to-face or telephone consultation about FPT with a gynaecologist in the Leiden University Medical Center (Leiden) or the Reinier de Graaf Hospital (Delft). Themes were communication about infertility and FPT, experience with the consultation on FPT, the decision making process, experience with FPT, feelings about possible infertility, and reflection on the local procedures. Of 46 eligible women, 12 (26%) declined. Of the 34 participating women, 21 had undergone FPT. Of the women who had had face-to-face consultations 47% underwent FPT vs. none of those who had had a telephone consultation. In retrospect, one in four women were dissatisfied with the timing of the consultation, but 85% were satisfied with the consultation. One-third indicated having felt a lack of clarity about the FPT options. Of those who underwent FPT, 75% were very satisfied with the procedures. Patients were generally satisfied with the procedures, but experience a difficult choice in which not all options appear to be clear. As centralization of FPT limits the options for face-to-face consultation, we have decided to develop a web-based decision-aid for FPT decision-making.

FERTILITY PRESERVATION OPTIONS FOR FEMALE PATIENTS: TWO YEARS OF EXPERIENCE.

D Manau, JM Calafell, Fabregues F, Guimera M, Civico S, Balasch J

Institut Clínic d´Obstetrícia, Ginecologia i Neonatologia. Hospital Clínic. Facultat de Medicina. Universitat de Barcelona. Institut d´Investigacions Biomèdiques August Pi i Sunyer ( IDIBAPS). Barcelona

The improvement in the survival of children and young women with cancer and another non oncologic conditions treated with gonadotoxic agents, has led to consideration of long-term side-effects of treatments. One of these effects is premature ovarian failure. Since 2005 patients were referred to our Unit from oncologist for aGnRH treatment. In 2007 we initiated a program of fertility cryopreservation. 57 patients were referred to the fertility cryopreservation program from different departments of our hospital (Oncology, Haematology and Autoimmune Diseases). A first evaluation included the following factors: age, pathology, type of chemotherapy, prognostic, parity, marital status and time available before the initiation therapy. After that, patients were informed about available strategies for fertility preservation: ovarian tissue freezing and embryo/oocyte cryopreservation. After conformity, we performed a gynaecological and ultrasound control, blood analyse and contact with the oncologist. A total of 57 patients were referred to our program . From this, 21 have been diagnosed with breast cancer, 15 with lymphoma, 7 with leukemia, 2 ovarian cancer, 7 with other types of cancer and 5 with autoimmune disorders. 7 women declined and 28 were excluded for different factors. The most frequent excluding factors were impossibility to delay cancer treatment, advanced age or health status. We performed 17 laparoscopies for cryopreservation of ovarian tissue, following biopsy (12) or unilateral oophorectomy (5). And 8 ovarian stimulated cycles were started for oocyte or embryo cryopreservation. The different fertility preservation treatments were well accepted for the patients (87%). Our results show the need for multidisciplinary treatment plan between gynaecologist and oncologist to increase patient acceptance and to choose the best option.

Cryopreservation of the whole sheep ovary—evaluation of the cryoprotectant DMSO by in vitro tests

Milenkovic M, Wallin A, Gharemani, M,, Brannstrom, M

Department of Obstetrics & Gynecology, Sahlgrenska Academy at University of Gothenburg, Sweden

Whole ovary cryopreservation and retransplantation of the ovary by vascular anastomosis has been suggested as a way to bypass the dramatic loss of follicles that occurs after avascular transplantation of cryopreserved ovarian tissue. The aim of the present study was to evaluate the effectiveness of the cryoprotectant dimethylsulphoxide (DMSO) to preserve the sheep ovary during freezing. Ovaries of sheep, 1–2 years of age, were primed with vaginal medroxyprogesterone for 12 days followed by equine CG (500 IU) 24 h before surgery to synchronize into follicular phase. During anaesthesia, a laparotomy was performed to isolate the ovary and a short vascular pedicle that included the ovarian artery and veins. The ovary was submerged in cold Ringer-Acetate, while the ovarian artery was cannulated with a 22/24 G over-the-needle Teflon catheter. Blood was flushed out from the ovary and either cryoprotectant solution (1.5 M DMSO, 0.1 M sucrose, 2% human serum albumin) or non-cryoprotecant solution (Ringer-Acetate, 0.1 M sucrose, 2% human serum albumin) was used for flushing (30 min at 80 mmHg pressure). Passive slow cooling was used and the ovaries were stored in liquid nitrogen. After thawing, ovaries were assessed with various test of viability (histology, in vitro perfusion, cultures of ovarian cells, live/dead assay). During in vitro perfusion DMSO-frozen ovaries responded with slightly higher progesterone and cAMP production than noncryoprotectant-frozen ovaries. In cell cultures DMSO-frozen ovaries showed higher progesterone production and this group also showed higher % of live cells both before and after perfusion. Histology indicated better tissue preservation after DMSO. This study demonstrates that several in vitro techniques can be used to assess the viability of an ovary that has been cryopreserved. The increased viability of the ovarian cells and the increased progesterone secretion from cells isolated form whole ovaries that have been cryopreserved in DMSO as compared to cryopreservation in ordinary Ringer Acetate points to the beneficial effects of cryoprotectants for whole ovary cryopreservation. Future studies will be aimed towards optimizing concentrations of DMSO, as well as freezing and thawing rates.

The safety of the vitrification technique using cryotop at our clinic

Kyono K, Nakajo Y, Nishinaka C, Shibuya Y, Sasaki K, Sakamoto E, Doshida M, Toya M

Kyono ART Clinic, Sendai, Japan

We did follow-ups on babies born following vitrification method using cryotop. 1) Cryopreservation methods were slow freezing (straw) and vitrification (cryotop). 2) One case received two vitrified-warmed blastocysts produced by ICSI using vitrified-warmed oocytes and frozen-thawed sperm were transferred. 3) We compared ordinary slow freezing using straw (straw group: 86 singletons) and vitrification using cryotop (top group: 257 singletons).in terms of the following measurements: the length of gestation periods, average birth weight, average height, average girths of chest and head, premature births, low birth-weight rates, stillborn rates, birth defect rates, and physical development up to the age of 6. We achieved a successful birth of a healthy boy following transfer of two vitrified-warmed blastocysts produced by ICSI using vitrified-warmed oocytes and frozen-thawed sperm in one case. The length of gestation period (week), average birth weight (g), average height (cm), average girths of chest and head (cm) in straw group vs cryotop group were all within the normal range. Premature births rates, low birth-weight rates, stillborn rates, and birth defect rates (straw group vs cryotop group) were 8.1% (17/86) vs 9.3% (24/257) , 16.2% (14/86) vs 9.3% (24/257), 0% (0/86) vs 0.8% (2/257) , and 2.2% (2/86) vs 1.2% (3/287), respectively. Physical development from birth to the age of 6 was within the normal range. In both straw and cryotop groups, physical development from birth to 6 years of age was normal. This suggests that the vitrification method using cryotop is simple, easy and safe, but further studies are still needed.

Sperm cryopreservation for patients with malignant or non-malignant diseases: the outcomes of 27 cases

Kyono K, Sato Y, Oota M, Nakajo Y, Takizawa T, Kyoya T, Hattori H, Kanto S.

Kyono ART Clinic, Sendai, Japan

We reviewed cases of sperm cryopreservation in malignant and non-malignant diseases. The clinical records of 27 cases from January 1997 to February 2008 were reviewed. The age at cryopreservation, marital status, diseases, timing of cryopreservation, semen quality, utility, duration of banking, and results were analyzed. The age at cryopreservation ranged from 18 to 51 years old. At the time of cryopreservation, 14 patients were married, 3 were engaged, and 10 were single. Their original diseases were testicular cancer in 13 patients, other solid tumors in 5, leukemia in 7, and collagen disease in 2. Eighteen patients had their sperm cryopreserved before gonadotoxic therapies and 9 after the therapies. Semen analysis results of 7 patients were within the normal range, and those of 19 were not, including 2 cases with azoospermia. The ones with abnormal semen results included 5 with testicular cancer, 5 with leukemia, and 3 with solid tumors. 6 patients used frozen/thawed sperm in ART, and 5 achieved pregnancies. Healthy babies were born in 4 cases. Two patients died. The periods of sperm banking ranged from 1 to 111 months. The candidates for sperm banking in this study varied compared to the previous reports. It is ideal to have sperm cryopreserved before spermatogenesis shows any decline after gonadotoxic therapy. Thus, it is very important to educate doctors in other fields as well as the public about these findings. Long term storage of sperm is extremely difficult in private clinics. We hope to get our government’s cooperation on management, control, supervision, charge, and other care regarding sperm banking.

Four successful pregnancies following assisted reproductive technology

Kyono K, Fukuda Y, Kuchiki M, Izumi M, Oota M, Sato Y, Doshida M, Toya M

Kyono ART Clinic, Sendai, Japan

We report 4 successful pregnancies of 4 women who had overcome their malignant diseases. The subjects were four female patients with acute lymphoblastic leukemia, aplastic aneamia, cervical carcinoma in situ, and breast cancer. The main measurements were their marital ages, disease onsets, the length of treatments and infertility periods, the number of retrieved oocytes, insemination technique, the number, and grade of transferred embryos, and whether there was any male factor. In case 1, the measurements were 36 years old, 37 years old, 3 years, 4 years, 2 oocytes, ICSI, 2 ETs, 1 embryo (D3, fresh 8 cells), and with male factor, respectively. In case 2, the measurements were 20 years old, 17 years old, 10 months, 5 years, 7 oocytes, ICSI, 2 ETs, 1 embryo (D5 frozen-thawed, 4BB), and with male factor, respectively. In case 3, the measurements were 28 years old, 31 and 33 years old, 14 months and 2 years, 9 years and 5 months, 7 oocytes, IVF, 2 ETs, 1 embryo (D5 frozen-thawed, 5BB), and without male factor, respectively. Finally, in case 4, the measurements were 31 years old, 39 years old, 2 years, 4 years, 4 oocytes, ICSI, 2 ETs, 1 embryo (D5 frozen-thawed, 4BC), and without male factor, respectively. We achieved successful pregnancy considering ovarian reserve after chemotherapy and male factors. It is very crucial to educate oncologists on relationships between aging and other factors such as ovarian reserve, pregnancy rate, and miscarriage rate. The fact that half of the infertility is caused by male factors, and pregnancy rate depends on types of cancer treatments. It is advisable to see fertility doctors and receive ART as soon as possible.

Effects of melatonin on vitrified testis transplants

Parichehr Pasbakhsh•, Masoud Hemadi, Aligholi Sobhani, Mehdi Abbasi, Gholamreza Hassanzadeh, Farid Abolhassani, Fardin Amidi.

•Department of Anatomy, Tehran University of Medical Sciences, Tehran, Iran.

Melatonin besides action on different physiological processes is an antioxidant and powerful direct free radical scavenger. This study assessed different concentration of melatonin on vitrified testis grafted into castrated mature mice. Testes from neonate (Balb/c) mice were vitrified-warmed and transplanted heterotopically into host. Following transplantation, melatonin (20, 50, 100, 200 mg/kg/day or saline) were applied via gavage to separate groups. Donor-specific IgM and IgG subtype antibodies, Th1 (IL-2 and IFN-γ) and Th2 cytokines (IL-4 and IL-10) and melatonin in the serum of recipients were measured using ELISA analyses. The subsequent survival (days 1–8 and 42) of the grafted testes were scored morphologically. Indices of cell proliferation and apoptosis within spermatogonia and interstitial cells of tissue grafts were determined by BrdU and TUNEL assay respectively. This study showed that with increasing doses of melatonin, the graft lifespan was significantly prolonged (P ≤ 0.05). Our results also, indicated that apoptosis in the spermatogonia was dramatically increased (P ≤ 0.05). Allospecific IgM and also IgG2a of recipients were reduced with increasing doses of melatonin (P ≤ 0.05). The average level of Th1 cytokines was significantly reduced in 200 mg/kg/day of melatonin (P ≤ 0.05). No significant differences in Th2 cytokines were observed with increasing melatonin. Also, the variable regimens of melatonin caused higher peak melatonin levels in serum after transplantation. These findings indicate a novel therapeutic approach, based on modulation of the immune and E/R through melatonin as a possible future immunosuppressant and antioxidant in organ transplantation.

Oocyte vitrification for fertility preservation in young women suffering from cancer.

Domingo J, Rabadán S, Martínez M, Tocino A, Cobo A, Pellicer A, García-Velasco JA.

Fundación IVI

Cancer survival rates have improved significantly over the last years. Consequences of cancer treatment involve ovarian failure and infertility. Several strategies have been proposed to develop a program for patients who wish to preserve their fertility before chemotherapy.

The efficacy of vitrification in clinical practice has been demonstrated in the past few years. Oocyte cryo-banking with the Cryotop vitrification method represents a viable option for healthy women, producing excellent survival rates and clinical outcome similar to those obtained with fresh oocytes. We present our experience with this method as useful tool for fertility preservation in cancer patients. During the last two years, fertility preservation was offered to 96 patients after their oncologist’s permission. Breast cancer was the most frequent tumor (n = 58; 60.4%), followed by Hodgkin lymphoma (n=13; 13.5%). Among these patients, 10.54% had previous children.. Oocyte vitrification (Cryotop method) was recommended on 79 of these patients after obtaining a written consent. The other options were ovarian tissue cryopreservation (n=2; 2%) or no treatment (n=15; 15.6%). In the oocyte vitrification group, ovarian stimulation was performed with a combination of letrozole 5 mg and mild stimulation (150 IU rFSH) under a GnRH antagonist protocol, and GnRH agonist to trigger final oocyte maturation when patients were diagnosed of breast cancer. For no hormonal-dependent cancers, stimulations were performed only with gonadotropins. Ovarian stimulation was started on the second or third day of the cycle or when serum E2 levels were under 60 pg/ml following a 3 mg GnRH antagonist injection. Letrozole was maintained until the return of menses. Mean age was 30 ± 8.9 years old. A total of 719 oocytes were retrieved (mean 9.4 ± 6.7). After evaluating nuclear maturity 536 (74.5%) mature oocytes were vitrified (mean 7.05 ± 5.08). The mean E2 level (262.5±190 pg/mL) in the letrozole group significantly lower when compared with the gonadotropins group (1,107 ± 743 pg/mL): p < 0.05. Oocyte vitrification for fertility preservation can be performed in people undergoing cancer treatment. Ovarian response in a mild stimulation cycle in these young patients seems to yield a good response and offers them the possibility of attempt a pregnancy with their own oocytes once cancer is cured.

Comparison between two techniques of follicular viability assessment for evaluation of a slow freezing procedure on human ovarian tissue

S. Sanfilippo1, M. Canis1,2,5, L. Ouchchane3, L. Janny1,4, JL. Pouly1,2,5, F. Brugnon1,4

1Université Clermont 1, UFR Médecine, EA 975, Laboratoire de biologie de la reproduction, Clermont-Ferrand, France.

2CHU Clermont-Ferrand, Gynécologie obstétrique, Hôtel Dieu, Clermont-Ferrand, France.

3Université Clermont 1, UFR

Médecine, Laboratoire de biostatistiques, Télématique et traitement d’image, Clermont-Ferrand, France. 4CHU Clermont-Ferrand, Biologie de la Reproduction, CECOS, Hôtel Dieu, Clermont-Ferrand, France. 5Centre International de Chirurgie

Human ovarian tissue cryopreservation is the best option for fertility preservation for patients undergoing anticancer treatment or suffering from pathologies potentially leading to premature ovarian failure. Follicular viability assessment allows the efficiency of the freezing/thawing procedure and the potential of viable follicles to be checked before grafting. The aim of our study was to adopt the more accurate test to evaluate human follicular viability, comparing the trypan blue and calcein AM/ethidum homodimer staining methods which are currently used in clinical research. Ovarian cortical slices were obtained from 10 consenting patients (mean 29 ± 1.8 SEM years) undergoing endoscopic surgery for benign cysts. Small follicles (<70 μm) were enzymatically isolated from fresh and frozen ovarian tissue by an original slow cooling protocol previously developed in our lab (Schubert et al., 2005). Dead and live follicles were identified with trypan versus calcein AM/ethidium homodimer (live/death® kit, Interchim, France) staining. Viability of at least 100 follicles was assessed per staining process using a light microscope for blue trypan staining or epifluorescent one for calcein AM/ethidium homodimer staining. The viability of each sample was assessed by 2 independent observers. Statistical analysis was carried out using a signedrank test for comparative analysis (p value < 0.05, significance level). Reliability was evaluated using intraclass correlation coefficient (ICC). Interobserver reliability assessed on fresh and thawed ovarian tissue was good for each staining method (Trypan blue: p = 0.12, ICC = 0.59; Calceine AM/ethidium homodimer: p = 0.27, ICC = 0.46). A significant systematic higher proportion of viable follicles was observed by using calcein AM/ethidium homodimer (+6.5 ± 1.9, mean ± SEM) compared with trypan blue (p = 0.001). The percentages of viable follicles before and after thawing did not differ statistically whether after trypan blue (18 ± 2.6% vs. 19 ± 2.5%; p = 0.70) or after calcein AM/ethidium homodimer staining (32 ± 3.7% vs. 25.2 ± 2.8%; p = 0.64). Our results suggest that blue trypan and calcein AM/ethidium homodimer staining are fast and appropriate methods for appreciation of ovarian quality in case of therapeutic cryopreservation. However, the systematic higher percentage of viable follicles after staining by calcein AM/ethidum homodimer compared with trypan blue meant that only data measured using the same method could be compared. The difference in staining specificities between these two methods could explain this systematic difference. The original slow freezing/thawing procedure used in this study appears to preserve follicular viability well, but other markers are needed to better assess the morphological and functional quality of ovarian tissues before grafting.

The Marketing of 'Fertility Insurance' in the United States and the Need for Greater Regulation of the Assisted Reproductive Technology Industry

Seema Mohapatra, JD, MPH

University of Central Florida School of Medicine

The multi-billion dollar Assisted Reproductive Technology ("ART") industry in the United States is largely unregulated and privately funded. The lack of regulation of this arena has resulted in women being exploited financially and emotionally by a self serving industry. This paper/presentation will explore the United States' dark history of exploitation in the medical research arena (examples: Tuskegee, Willowbrook, Kennedy Krieger). Given this backdrop and the financial conflicts of interests facing fertility clinics and specialists, it is overly optimistic and naïve to expect the ART industry to act in the best interests of their clients. This paper/presentation examines the example of oocyte cryopreservation, commonly known as egg freezing, to demonstrate the need for greater regulation in the ART arena. In the past, only female cancer patients who had to undergo chemotherapy and therefore risk future infertility have had access to egg freezing. However, in the United States, this procedure is increasingly being offered to healthy women in their late twenties and early thirties as a method of extending their fertility. Theoretically, this technology sounds promising as a means for allowing women to have greater control over their biological clocks. Unfortunately, there are only a few examples in the scientific literature to support the contention that egg freezing will result in a successful pregnancy or live birth. This paper/presentation examines how the unregulated marketing of this largely untested procedure to a vulnerable population, women in their thirties who have strong feelings about fertility, has great potential for exploitation. Although many have argued that this lack of regulation has many benefits (allowed for greater technological innovations in the area of assisted reproduction and allowed women to have more freedom over their reproductive choices), this paper will suggest how regulation may actually increase autonomy.

Protective effects of melatonin on hetrotopical transplanted vitrified-warmed mice testis tissue.

Aligholi Sobhani•, Masoud Hemadi, Parichehr Pasbakhsh, Mehdi Abbasi, Gholamreza Hassanzadeh, Farid Abolhassani, Fardin Amidi.

•Address for Correspondence: Department of Anatomy, Tehran University of Medical Sciences, Tehran, Iran.

Cancer therapy applied to young patients most often has severe effects upon male fertility. Present study investigated the protective role of melatonin on neonate mouse testis tissue during vitrification-thawing and heterotopic transplantation into castrated recipient mice.

Materials and methods: Vitrified testes from neonate inbred mice, candidates for transplantation were thawed under standard conditions with or without the addition of 100 µM melatonin, respectively. Following transplantation, melatonin (20 mg/kg/day) or saline solution was applied as gavage to the treated and the non- treated groups (control) respectively. In 1–8 and 42 days Melatonin, gonadothropins and steroids concentrations were evaluated by ELISA. Spermatogonia survival and spermatogenesis were also assessed by TUNEL and Brdu respectively. Histological and immunohistochemical studies showed that melatonin could improve the spermatogonia quality in the testis graft. Plasma LH and FSH levels were significantly (P≤0.05) higher in the castrated host than intact mice (P≤0.05) at before grafting. However, the melatonin administration reduced these hormones into nearly similar concentrations to those in intact mice. The correlation coefficients between gonadotropins and melatonin concentrations during the days of transplantation of the testis grafts were significantly different from zero (P≤0.05). However, testosterone secretions were not affected by melatonin treatment significantly. The results of present study suggest that melatonin has a positive effect on testis tissue after vitrification, thawing and heterotopic transplantation. Also, it could be beneficial on hypothalamic- pituitary–testis axis of the recipient castrated mice.

Preservation of ovarian function following ovarian cryopreservation with GnRH analogue co-treatment

Suguru Igarashi¹, Nao Suzuki¹, Shu Hashimoto², Seido Takae¹, Yoshiharu Morimoto², Bunpei Ishizuka¹

¹Department of Obstetrics and Gynecology, St. Marianna University, School of Medicine, Kanagawa, Japan ²IVF Namba Clinic, Osaka, Japan

In recent years, the number of young women with uterine cancer or breast cancer has been increasing. For these patients who underwent surgery or performed chemotherapy, it is very important to preserve ovarian function and keep the quality of life as one woman. Recently it has been published that the techniques of cryopreservation and transplantation of the ovarian cortex, and children have been born after successful orthotopic autotransplantation into the residual ovaries. One of the methods for preventing ovarian damage is the administration of GnRH analogue during or prior to chemotherapy. In this study, we compare the ovarian function with the GnRH analogue administration before cryopreservation and transplantation of the ovarian cortex. Materials and Methods: Six to eight weeks of Wister rats were divided into 3 groups such as control, vitrification group, and slow-freezing group. After cryopreservation and transplantation, we confirmed the day of the recovery of estrous cycle. Then, we examined the same 3 groups which were administrated with GnRH analogue (3.75 mg/kg) before cryopreservation and transplantation, and confirmed the recovery of estrous cycle days.

Results: Ovarian function of all groups was preserved, and ovarian function of all the other groups even using GnRH analogue was preserved. Vitrification method is more cheap and convenient rather than slow-freezing method. Therefore, vitrification method might be standard method of cryopreservation and transplantation of the ovary. Since the ovarian function was also preserved by administrating GnRH analogue, this method should be effective for the young cancer patients who already performed chemotherapy

Reoxygenation and revascularization processes of human ovarian xenotransplants

Van Eyck, AS.; Dolmans, MM.; Van Langendonckt, A.

Gynaecology research unit, Université Catholique de Louvain, Brussels, Belgium

The success of ovarian tissue transplantation appears to be limited by ischemia-reperfusion damage caused by an avascular procedure. Our aim was to characterize the oxygen microenvironment and revascularization process ovarian xenograft in the early post-grafting period. Nude mice were intraperitoneally grafted with frozen-thawed human ovarian tissue obtained from 6 patients. Graft reoxygenation was assessed by electron paramagnetic resonance (EPR), which allowed non-invasive and repeated measurement of partial oxygen pressure (pO₂). The pO₂ was monitored on post-grafting days 3, 5, 7, 10, 14, 17 and 21 (n mice=18) and graft perfusion was spatially evaluated by Hoechst 33342 uptake on days 3, 5 and 10 (n mice=18). Murine and human vascularization was analyzed by CD31 and von Willebrand factor double staining. ANOVA or t-tests where applied for statistical analysis where appropriate. Results were expressed as mean ± SEM. P < 0.05 was considered statistically significant. Before day 5, a period of hypoxia was identified (day 3 pO₂:13.3 ± 7.4 mmHg). Fragments were largely non-perfused and occasional murine neovessels were located at the periphery. From day 5, gradual but significant reoxygenation was observed over the subsequent 5 days (day 5 pO₂: 22.3 ± 11.6 mmHg, p < 0.001; reoxygenation speed: slope ^D5-D10 = 2.4–0.4 mmHg/d, p < 0.001). Murine neovessels progressively penetrated from the periphery and were colocalized with perfused areas. By day 10, the increase in perfusion and murine vascularization was significant. The center of the fragments was perfused and a significant increase in human vasculature was observed. On day 21, pO₂ values were found to have stabilized and remained higher than the initial hypoxic value (day 21 pO₂: 26.2–7.3 mm Hg; p < 0.001). After an initial hypoxic and avascular period, murine angiogenesis initiated reoxygenation and reperfusion of the graft from day 5 onwards. By day 10, human vessels participated in graft revascularization. Host and graft vessels contributed sequentially to graft revascularization and are therefore potential targets to reduce the ischemic period after grafting.

L-Carnitine improves fertilization rate from mouse vitrificated-thawed oocytes

Wu, H-H.^1,2, Tsai, H-D., Chang, M-H., Wu, C-H., Yeh, G-P. and Yang, J-G.

Changhua Christian Hospital, Changhua, TAIWAN; 2. Chung Shan Medical University, Taichung, TAIWAN

L-Carnitine (LC), an important water-soluble molecular in fat metabolism, can protect cell membrane and DNA from free oxygen radicals damage by removing superoxide anion and inhibiting lipid peroxidation. Previous studies showed that embryo culture medium supplementation with L-Carnitine may improve embryogenesis of cultured embryos. This study is to evaluate the effect of L-Carnitine supplement on embryo development of vitrificated-thawed mouse oocytes. This is an animal experimental study in reproductive research center at a tertiary medical center. There are four groups in the study: Group A, 44 fresh oocytes cultured in medium without LC; Group B, 49 fresh oocytes cultured with 0.3 mg/mL LC; Group C, 117 vitrificated oocytes cultured without LC; Group D; 69 vitrificated oocytes cultured with 0.3 mg/mL LC. Vitrificated oocytes were cryopreserved by vitrification using CryoTop®, and were thawed 1 week later. All oocytes were fertilized in vitro with sperms from male mice in Quinn's Advantage® Fertilization Medium. Embryos were assessed every day after insemination. On the day after insemination, fertilized embryos will develop to two-cell stage. Proportional variables were compared using the Fisher’s exact testt. All statistical tests were based on two-tailed probability. P value < 0.05 was considered statistically significant. Vitrificated oocytes had low fertilization rate compared with fresh oocytes [59% (69/117) vs 96% (42/44); odds ratio 0.07 (0.01–0.29), p-value < 0.05]. When cultured with L-Carnitine, the difference was not statistically different between vitrificated oocytes and fresh oocytes [81% (50/62) vs 86% (42/49); odds ratio 0.69 (0.21–2.13), p-value > 0.05]. From vitrificated oocytes, the add of L-Carnitine in culture medium improved fertilization rate [81% (50/62) vs 59% (69/117); odds ratio 2.9 (1.34–6.6), p-value < 0.05]. In our study, the supplement of L-Carnitine in culture medium can improve fertilization rate in the vitrificated-thawed oocytes. The supplement of L-Carnitine in culture medium may decrease oxidative stress, therefore may increase fertilization rate. However, it needs more experimental studies to prove its effect.

Anti-mullerian hormone (AMH) reflects ovarian reserve over one-year after intraovarian infusion of sphingosine-1-phosphate (S1P) and S1P agonist, FTY720 (FTY), prior to ovarian X-irradiation in macaques

Zelinski, M.B.¹; Toscano, N.P.²; Winger-Schorr, E. ¹; Murphy, M.M. ³; Jacob, D.¹; Fanton, J.¹; Lawson, M.S. ¹; Tilly, J.L. ⁴

¹Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, Oregon, USA; ²Centro de Educacion Medica e Investigaciones Clinicas, Buenos Aires, Argentina; ³Pacific Northwest National Laboratory, Richland, Washington, USA; ⁴Vincent Center for Reproductive Biology, Harvard Medical School, Boston, Massachusetts, USA

Intraovarian delivery of the anti-apoptotic agents, S1P and FTY, can protect a cohort of ovarian follicles from radiation-induced damage as well as allow fertility in macaques, and offers a novel option for fertility preservation. The duration for fertility potential following ovarian protection in primates is uncertain. This study determined whether circulating AMH levels reflect ovarian reserve after in vivo X-irradiation of ovaries pre-treated S1P or FTY. Rhesus monkeys received intraovarian infusion via osmotic minipumps with vehicle (V; n = 9), S1P (n = 9) or FTY ( n = 6) into both ovaries for 7 days. Animals a) underwent unilateral ovariectomy (ovx) prior to ovarian sham (n = 3, V + Osh) or X-irradiation (15 Gy; n = 3, V + OXI; n = 6, S1P + OXI; n = 3, FTY + OXI) of the other ovary which was removed up to 12 mo later; or b) received bilateral V + OSh (n = 3), S1P + OXI (n = 3) or FTY + OXI (n = 3) with bilateral ovx 21 mo later. Serum levels of AMH were measured in pre- and post-treatment cycles using ELISA (Diagnostic Systems Laboratories, Webster, Texas, USA). AMH levels were similar in all groups before treatment (range 2.0 to 3.5 ng/ml), and were maintained in V + Osh during the 21-mo post-treatment interval. AMH levels were nondetectable in V + OXI post-treatment, wherein preantral follicles were absent. In macaques with 1 ovary, AMH levels were <0.5 mg/ml up to 12 mo post-S1P + OXI, and slowly increased in animals with 2 ovaries from 0.8 ± 0.2 to 1.4 ± 0.2 ng/ml between 12 and 21 mos, respectively. AMH levels were consistently greater (P < 0.05) in FTY + OXI relative to S1P + OXI; levels in animals with 1 ovary were 0.4 ± 0.1 1 ng/ml. In FTY + OXI with 2 ovaries, AMH levels (1.8 ± 0.2 ng/ml) did not change between 12–21 mos post-radiation, but remained lower (P < 0.05) than V + Osh. AMH levels correlated with the cohort of primordial follicles remaining after V + OSh (85±2%) and FTY + OXI (22 ± 9%) which was greater (P < 0.05) than S1P + OXI (1±0.3%). Circulating levels of AMH correlated with the remaining cohort of primordial follicles that were protected from radiation-induced damage by intraovarian infusion of S1P and FTY. Thus, AMH can be a reliable indicator of ovarian reserve up to 21 mos after in vivo X-irradiation of ovaries pre-treated with anti-apoptotic agents. Whether ovarian reserve and fertility are maintained for longer intervals post-radiation of ovaries protected with anti-apoptotic agents in vivo remains to be determined in primates.

Childhood cancer treatment causes less severe gonadotoxicity than adult cancer treatment: A retrospective study.

Zervomanolakis¹, S. Foeger², M.S. Reifer², L. Wildt².

¹Mitera General, Maternity & Children’s Hospital, IVF-Unit, Athens, Greece.

²University Clinic of Gynecological Endocrinology, Innsbruck Medical University, Austria.

During the last three decades the survival rate of young women undergoing chemotherapy or radiation because of cancer has raised in response to the improvement of the chemotherapeutical regimes. Nevertheless, little attention has been paid to the effect of cytotoxic therapy on these patients in the past. Objective of our study is to examine the effects of cytotoxic therapy on fertility preservation regarding patients` age during treatment. 75 patients of a current age between 18 and 50 years, who have undergone chemotherapy and/or radiation due to leukemia, sarcomas or lymphomas in the last 15 years in our university clinic, were included in the retrospective study. 30 patients were under 18 years during treatment (group A). All patients of the group A received chemotherapy, while 14 patients (46,66%) were treated with both chemotherapy and radiation. 45 patients were adults during treatment (group B). All patients of the group B received chemotherapy, while 23 patients (51,2%) were treated with both chemotherapy and radiation. Amenorrhea was defined as absence of menstruation for at least 12 months after end of treatment. Anti-müllerian hormone (AMH), as a marker of ovarian reserve, gonadotropins, hot flushes without contraceptive pills or hormonal replacement therapy, fertility preservation methods and pregnancies after treatment were evaluated. Group A had a significantly higher AMH level (3.33 ± 3.26 μg/l vs. 1,07 ± 0,89 μg/l , p < 0,05). Follicle stimulating hormone (FSH) was significantly lower in the group A (5,69 ± 3,2 IU/l vs. 56,25 ± 1,63 IU/l, p < 0,05), indicating premenopausal status. The rate of amenorrhea without contraceptive pills or hormonal replacement therapy was significantly higher in the group A (23,1% vs. 80,0%, p = 0,03), while the rate of patients with hot flushes (8,3 %, vs. 64,3%, p < 0,05) was significantly lower in the group A. Although none of the patients of the group A experienced cryopreservation of ovarian tissue or treatment with GnRH analogues as fertility preservation methods compared with the group B, in which 26,7% of the patients was treated with GnRH analogues and 22,2% of the patients underwent a cryopreservation of ovarian tissue, 13,3% of the patients in the group A conceived after cytotoxic treatment compared with 4,5% of the patients in the group B (p = 0.39). Our study provides evidence that childhood cancer treatment causes less severe gonadotoxicity than adult cancer treatment. Patients with cancer need to be informed about the effects of cytotoxic therapy on their fertility and be offered all possibilities to preserve their ovarian function.

POPULATION STUDY ON FREQUENCY AND OUTCOME OF PREGNANCY IN WOMEN AFFECTED BY NEOPLASIA IN THE VENETO REGION (North-East Italy)

Cesare ROMAGNOLO M.D. (1,2), Tiziano MAGGINO M.D.(3), Paola ZAMBON M.D. (4)

(1)Head Gynecologic Oncology Unit - “Dell'Angelo” Hospital Mestre—Venezia (Italy); (2) Presenting Author; (3) Head Gynecology and Obstetrics Department “Dell'Angelo” Hospital Mestre—Venezia (Italy); (4) Veneto Tumor Registry - Department Oncological Science - Veneto Institute of Oncology- IRCS- Padova (Italy)

To evaluate the frequency and the outcome of pregnancy in women affected by cancer we carried out a descriptive analysis using the data base of the Veneto Tumour Registry (RTV) which has been active since 1987 and covers about half of the population of Veneto region (North East Italy, 4.527,694 inhabitants at the 2001 census). MATHERIAL AND METHODS: We have extracted from the database of the RTV the women aged 15–49 years with diagnosis of cancer between 1997 and 2001; we have considered all sites of tumour, except non melanoma skin cancer. To retrieve cases with a pregnancy, we have used the hospital discharges for the period 1997–2005, with the International Classification of Diseases codes, ninth edition (ICD IX), in the principal diagnosis: pregnancy, puerperium, delivery. As a results of this cross-linkage we have obtained a cohort of 189 women, 15–49 year old, who represents the study population we report. RESULTS: In the period 1997–2001, 3,872 women aged 15–49 in the population covered by RTV have had a diagnosis of malignant tumour, 14% of all ages. In this cohort, 189 (5%) had at least one pregnancy after their neoplasia diagnosis and before 31st December 2005. The median age was 31 (interquartile range 25–34). In this population we observed 186 deliveries, 29 spontaneous abortion and 28 volunteer miscarriages. One hundred thirty seven women out of 189 have had only pregnancies; 31 have had only miscarriage; 21 have had both pregnancies and miscarriages.. In addition, 48 women have had more than one pregnancy and 28 women have had more than one delivery. No stillbirth have been reported. In the first year the fertility rate for women under study is 0.4 versus a constant rate of 1.2 in the general population. In the following years it grows steadily and becomes quite constant after the 6° year with a level of 0.6 that is one half of that of the general population. The TFI calculated from our study population demonstrates that pregnancy effectively occurs in many cases after neoplasia’s diagnosis and treatment, but in the same time we have to question us if the small value of TFI after ten years may be considered, at least in some patients, a failure of our efforts to preserve fertility in our patients.

Overcoming infertility by ZIFT and IVF: a clinical case study

Authors: Ghavami Adel, Hadye; Kosarian, Maryam

IVF and ZIFT are two methods for achieving successful pregnancy. We employed these methods, respectively, to study over 120 infertile women from June 2008 to January 2009. We divided our patients into two different age groups, 77 patients below the age of 35 (group A), and

43 of them above 35 years old (group B). ZIFT was used for 82 patients, among them 55 were belong to group A and 27 patients were belong to group B. The results indicate 63.3% of success in patients below 35 years old, while we succeeded around 52% of pregnancy in patients above 35 years old. IVF is also used for process of conceiving in 38 patients, 22 patients below 35 years old and the rest above, respectively. The younger group shows 45.5% successful pregnancy, whereas only 37.5% positive response has achieved in patients above 35. The above data suggests that at our clinic patients who have had multiple failed IVF cycles or who have patent tubes and they are older should consider ZIFT as a treatment option. Estradiol valerate was prescribed for 74 patients in groups A and B, in 62 patients who used estradiol valerate (1–2 mg) in their first and second period of cycle (days 1–7 and days 7–12) in group A, 63.6% pregnancy was achieved; whereas 58.6% positive response was found in group B. In another case in 12 patients from group A who were prescribed estradiol valerate in their second period of cycle, we achieved 50% success. These results indicate that patients who used estradiol valerate in their first and second cycles show a better results compared to the patients who received the same dose in their second cycle.

PRESERVATION OF MEN’S FERTILITY IN PATIENTS WITH LYMPHOMA.

Kiseleva M., Konov A., Bashkatov S., Karpeikina M..

Medical Radiological Research Center RAMS

249036 ul.Koroleva b.4, Obninsk, Kaluga reg., Russia

There are 2.6 millions cancer patients in the Russian Federation. Among them there are about 50% reproductive-aged men and women. The cancer treatment (heavy chemotherapy and/or radiotherapy) may reduce fertility by damage of ovaries and testicles. For these patients, there are several options that may help to preserve fertility before and after cancer treatment. If the patient has a partner or accepts donor sperm, embryo cryopreservation should be considered first. If not for preserving men’s fertility can be used cryopreservation of sperm or testicles tissue. For the women of reproductive age with cancer there are special options like ovarian tissue and eggs cryopreservation, also ovarian transposition out of the irradiation field before radiotherapy. Because of the variations in type and dose of chemotherapy, the type of cancer, the time available prior to onset of treatment, the patient’s age and the partner status, each case is unique and requires a different strategy of fertility preservation. We collected the semen samples of 21 patients (from 16 to 34 years of age) with Hodgkin’s and non-Hodgkin’s lymphoma before starting chemotherapy or radiation therapy and frozen at sperm bank. Semen samples were cryopreserved as follows: each aliquot was diluted in the same volume of cryoprotectant medium consisting of 15% glycerin, 2% sucrose, 0.4% human serum albumin in aqueous solution. Cryoprotectant medium was added sequentially drop by drop, with constant mixing. Each aliquot was then placed in cryotubes, frozen and transferred to a liquid nitrogen tank, until use. Samples can be stored for years and used later for insemination. A total of 21 sperms from patients with Hodgkin’s and non-Hodgkin’s disease were collected, analyzed and frozen. Our results indicate that in 16/21 cases (76,19%) were astenozoospermia, in 12/21 cases (57,14%) were olygozoospermia and only 5/21 cases (23,8%) were normozoospermia. We are establishing the first human sperm bank for cancer patients in the Russian Federation. Fertility preservation should be an integral part of improving the quality of live in cancer survivors; however, it is neither possible nor ethical to recommend the same recipe for every cancer patient. The first goal is to cure the cancer, even if the treatment causes sterility.