Full-term newborn after repeated ovarian tissue transplants in a patient treated for Ewing sarcoma by sterilizing pelvic irradiation and chemotherapy.

We report the first successful transplantation of cryopreserved ovarian cortical tissue into heavily irradiated tissues in a patient who had received sterilizing pelvic radiotherapy (54 Gy) and 40 weeks of intensive high-dose chemotherapy for the treatment of Ewing's sarcoma 14 years earlier. Repeated transplantation procedures were required to obtain fully functional follicular development. Enlargement of the transplants over time and increase of the size of the uterus were demonstrated on sequential ultrasonographic examinations. Eggs of good quality that could be fertilized in vitro were obtained only after a substantial incremental increase of the amount of ovarian tissue transplanted. Single embryo replacement resulted in a normal pregnancy and the birth of a healthy child by cesarean section at full-term. No neonatal or maternal postoperative complications occurred. Women facing high-dose pelvic radiotherapy should not be systematically excluded from fertility preservation options, as is currently the trend.

Preservation of human ovarian follicles within tissue frozen by vitrification in a xeno-free closed system using only ethylene glycol as a permeating cryoprotectant.

OBJECTIVE: To study the preservation of follicles within ovarian tissue vitrified using only one or a combination of three permeating cryoprotectants. DESIGN: Experimental study. SETTING: University hospital. DONOR(S): Ovarian tissue was donated by consenting women undergoing elective cesarean section. INTERVENTION(S): Ovarian tissue was vitrified in closed sealed vials using either a combination of dimethyl sulfoxide, 1,2-propanediol, and ethylene glycol (EG), or only EG as permeating cryoprotectants. MAIN OUTCOME MEASURE(S): Ovarian tissue was vitrified with the use of two vitrification methods. Tissue from the same donor was used for comparison of two different solutions. The morphology of the follicles was evaluated after vitrification, warming, and culture by light microscopy and transmission electron microscopy. Apoptosis was assessed by immunohistochemistry for active caspase-3 in fresh and vitrified tissue. RESULT(S): Light and electron microscopic analysis showed equally well preserved morphology of oocytes, granulosa cells, and ovarian stroma when either of the vitrification solutions was used. No apoptosis was observed in primordial and primary follicles. CONCLUSION(S): Using only EG as a permeating cryoprotectant in a closed tube gives as good ultrastructural preservation of ovarian follicles as a more complicated system using several cryoprotectants.

Apoptosis of human ovarian tissue is not increased by either vitrification or rapid cooling.

This study evaluated the incidence of morphological changes, as assessed by light microscopy, and apoptosis in vitrified and rapidly cooled human ovarian tissue. Apoptosis was assessed 30 min and 24h after warming using transmission electron microscopy, terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and DNA fragmentation, as determined by gel electrophoresis. The results showed no significant changes in morphology, chromatin condensation, DNA fragmentation or TUNEL-positive cells in follicles attributable to cryopreservation or exposure to the cryoprotectant solutions alone. In conclusion, the cryopreservation protocols did not affect the incidence of apoptosis and either protocol could be an alternative to slow cooling of ovarian tissue. This study evaluated the incidence of morphological changes, as assessed by light microscopy, and apoptosis in human ovarian tissue cryopreserved using two different methods, i.e. vitrification and rapid cooling. Apoptosis was assessed in tissue 30 min and 24h after warming using transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and DNA fragmentation as determined by gel electrophoresis. The results showed no significant changes in morphology, chromatin condensation, DNA fragmentation or TUNEL-positive cells in follicles attributable to cryopreservation or exposure to the cryopreservation solutions alone. In conclusion, the cryopreservation protocols did not affect the incidence of apoptosis in human ovarian tissue and either protocol could be an alternative to slow cooling for the preservation of ovarian tissue.

Clinical grade vitrification of human ovarian tissue: an ultrastructural analysis of follicles and stroma in vitrified tissue.

BACKGROUND: Cancer therapy is one of many conditions which may diminish the ovarian reserve. Banking of human ovarian tissue has become an option for the preservation of female fertility. We have shown that vitrification is an excellent method to cryopreserve ovarian tissue. To carry out vitrification in a clinical setting, we have developed a clinical grade closed system to avoid direct contact of ovarian tissue with liquid nitrogen. METHODS: Ovarian tissue was obtained by biopsy from 12 consenting women undergoing Caesarean section. Tissues were vitrified in cryotubes, using dimethyl sulphoxide, 1,2-propanediol, ethylene glycol and polyvinylpyrrolidon as cryoprotectants. Non-vitrified and warmed-vitrified tissue was compared by light and electron microscopic morphology of the follicles within the tissues. RESULTS: We did not see any differences in the light or electron microscopic ultrastructure of oocytes between non-vitrified and vitrified tissues. No irreversible subcellular alterations in vitrified tissues were seen. CONCLUSIONS: The ultrastructure of follicles within the vitrified human ovarian tissue was well preserved using cryotube in a closed vitrification system to avoid direct contact of liquid nitrogen. The system is compatible with the European tissue directive.

Cytologic screening and human papilloma virus test in women undergoing artificial fertilization.

BACKGROUND: Infertile women in Sweden are offered in vitro fertilization (IVF) within the frame of the social security system. The present investigation was undertaken to evaluate the prevalence of genital human papilloma virus (HPV) infection in relation to the results of cytologic screening and to the infertility in these women. MATERIAL AND METHODS: Two hundred and fourteen women, mean age 32 years (range 20-40), admitted to the Center for Reproduction at Uppsala University Hospital for investigation of infertility and IVF were studied. Human papilloma virus tests were performed by a sensitive polymerase chain reaction-based technique in cervical smears obtained at a medical examination or during oocyte retrieval. Cytologic screening results were obtained from medical records or at the time of investigation. The infertile women were compared with 197 healthy female controls. RESULTS: Infertility resulted from female factors in 47% and male factors in 29% of the cases, and remained unexplained in 24%. Seven percent of the infertile women were HPV-positive compared with 9.1% of the controls. Only genital and oncogenic HPV types were identified. Human papilloma virus type 16 was most prevalent, and examination of the HPV 16 E6 gene showed that this prototype predominated over variants. No correlation was found between HPV infection and cause of infertility. Abnormal cytology was observed in 2.3% of the infertile women and 4.1% of the controls. CONCLUSIONS: Human papilloma virus infections might appear somewhat less frequently in infertile women admitted for IVF than in a control population. In both groups HPV infection was more common than cytologic abnormalities, possibly indicating that present HPV tests are more sensitive in detecting HPV infections than cytologic screening.

Follicles are found in the ovaries of adolescent girls with Turner's syndrome.

Infertility caused by ovarian failure is a characteristic feature in Turner's syndrome. Spontaneous pregnancies are seen in 2-5% of these women, and up to 30% have at least some pubertal development, indicating the presence of follicles in their ovaries in adolescence. It has not been clear at which age the follicles disappear. We analyzed the numbers and densities of follicles in ovarian cortical tissue from nine adolescent girls with Turner's syndrome who came to our clinics after having been informed about the study, with an aim to preserve ovarian tissue for possible infertility treatment later in life. A quarter to one whole ovary was laparoscopically removed for the procedure. Follicles were seen in the biopsy tissue in eight of nine subjects from whom ovarian tissue was laparoscopically obtained, the highest numbers being seen in the youngest girls and in those with mosaicism. In one 17-yr-old girl, no ovarian tissue was found. Follicle density was correlated with serum levels of FSH; individuals with the lowest FSH levels had the highest follicle density. One to 190 follicles were counted in the approximately 0.1-2.0 mm(3) of tissue analyzed, giving a density of 1.5-499 follicles/mm(3) of ovarian cortical tissue. Girls up to the age of 17 had primordial follicles in their ovaries. Three girls, two aged 15 yr and one aged 19, had only secondary follicles, with many being atretic. Our finding that adolescent girls with Turner's syndrome still have follicles in their ovarian cortical tissue raises the possibility of future fertility through cryopreservation of ovarian tissue. However, before such procedures can be recommended for clinical management, it is essential that future studies be performed to determine whether the oocytes retrieved from girls with Turner's syndrome have a normal chromosomal complement.

Embryo transfer after 2 or 5 days of IVF culture: a retrospective comparison.

BACKGROUND: To determine whether prolongation of embryo culture in vitro from day 2 to day 5 after ovum pick-up (OPU) and fertilization can improve the results of in vitro fertilization (IVF), and the morphology of the spare embryos on day 2 can predict the developmental capacity during prolonged culture. We also wanted to consider this as a strategy to avoid twin pregnancies if it could be possible to transfer only one blastocyst at a time in the future. METHODS: A retrospective analysis with embryo transfer timed according to the weekday of OPU. Embryo transfer was performed on day 2 in 103 cases and on day 5 in 120 cases. Only one cycle per couple was included. RESULTS: The pregnancy rates per embryo transfer on day 2 (27/103, 26%) and day 5 (36/120, 30%) were similar. There were significantly more miscarriages in the day 5 (50%) than in the day 2 group (22%, p = 0.02), but there was no significant difference in the baby take home rate (20% in day 2 group, 15% in day 5 group). The morphological appearance of the embryos on day 2 was poorly correlated to the developmental potential during prolonged culture in vitro. On day 5, transfer of one or two blastocysts resulted in a pregnancy rate that tended to be higher than that after transfer of morulae only. CONCLUSION: Prolongation of embryo culture from day 2 to day 5 did not improve the clinical outcome of the IVF treatment when measured as baby take home rate. Therefore, for the time being, this strategy does not increase our chances to move towards single embryo transfer.