How can fertility counseling be implemented for every newly diagnosed pediatric patient facing gonadotoxic treatment?-A single-center experience.

Since the survival rates of pediatric patients undergoing cancer treatment or hematopoietic stem cell transplantation (HSCT) have increased rapidly in recent decades, the late effects of treatment are now an important focus of patient care. Access to fertility preservation (FP) procedures as well as their financing differs considerably across Europe. However, some countries in Europe have recently changed the legal basis for financing FP procedures; therefore, the implementation of structures is mandatory to give patients access to FP. In this prospective cohort study, we characterized the process for establishing pediatric fertility counseling, including the development of an in-house standard procedure for recommendations regarding FP with potentially gonadotoxic treatment and valuating data from all FP counseling sessions. All data concerning patient characteristics (pubertal status, disease group) and recommendation of FP measures were prospectively collected and adoption of FP measures analyzed. Prior to the establishment of a structured process for FP in our pediatric oncology and stem cell transplantation center, there was no standardized FP counseling. We demonstrate that with the establishment of an inhouse standard procedure, it is possible to give consistent yet individualized FP counseling to approximately 90% of our patients facing gonadotoxic treatment, counseling over 200 patients between 2017 and 2019. This pilot study could potentially be adapted in other pediatric hematology, oncology, and stem cell transplantation centers to allow a more standardized handling of FP counseling for all patients facing gonadotoxic treatment.

A novel mathematical model of true ovarian reserve assessment based on predicted probability of poor ovarian response: a retrospective cohort study.

PURPOSE: To establish a mathematical model for assessing the true ovarian reserve based on the predicted probability of poor ovarian response (POR). METHODS: In this retrospective cohort study, a total of 1523 GnRH-antagonist cycles in 2017 were firstly analyzed. The ovarian responses were calculated based on the number of retrieved oocytes. The continuous variables were converted into categorical variables according to cutoff values generated by the decision tree method. The optimal model was identified using forward stepwise multiple logistic regression with 5-fold cross-validation and further verified its performances using outer validation data. RESULTS: The predictors in our model were anti-Müllerian hormone (AMH), antral follicle counts (AFC), basal follicle-stimulating hormone (FSH), and age, in order of their significance, named AAFA model. The AUC, sensitivity, specificity, positive predictive value, and negative predictive value of AAFA model in inner validation and outer validation data were 0.861 and 0.850, 0.603 and 0.519, 0.917 and 0.930, 0.655 and 0.570, and 0.899 and 0.915. Ovarian reserve of 16 subgroups was further ranked according to the predicted probability of POR and further divided into 4 groups of A-D using clustering analysis. The incidence of POR in the four groups was 0.038 (0.030-0.046), 0.139 (0.101-0.177), 0.362 (0.308-0.415), and 0.571 (0.525-0.616), respectively. The order of ovarian reserve from adequate to poor followed the order of A to D. CONCLUSION: We have established an easy applicable AAFA model for assessing true ovarian reserve and may have important implications in both infertile women and general reproductive women in Chinese or Asian population.

Dexamethasone does not prevent malignant cell reintroduction in leukemia patients undergoing ovarian transplant: risk assessment of leukemic cell transmission by a xenograft model.

STUDY QUESTION: Does dexamethasone (DXM) incubation avoid the reintroduction of leukemic malignant cells after ovarian tissue retransplantation in vivo? SUMMARY ANSWER: DXM incubation prior to retransplantation of ovarian tissue does not prevent reintroduction of leukemic cells. WHAT IS KNOWN ALREADY: Retransplantation of cryopreserved ovarian cortex from patients diagnosed with acute lymphoblastic leukemia (ALL) involves a risk of reintroducing malignant cells. DXM treatment is effective at inducing leukemic cell death in vitro. STUDY DESIGN, SIZE, DURATION: This was an experimental study where ovarian cortex fragments from patients with ALL were randomly allocated to incubation with or without DXM (n = 11/group) and grafted to 22 immunodeficient mice for 6 months. In a parallel experiment, 22 immunodeficient mice were injected i.p. with varying amounts of RCH-ACV ALL cells (human leukemia cell line) and maintained for 4 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cryopreserved ovarian fragments from patients with ALL were exposed in vitro to 0.4 µM DXM or basal media (control) prior to xenograft into ovariectomized severe combined immunodeficiency (SCID) mice (experiment 1). After 6 months of monitoring, leukemia cell contamination was assessed in ovarian grafts and mouse organs by histology, PCR (presence of mouse mtDNA and absence of p53 were together considered a negative result for the presence of human cells) and detection of immunoglobulin monoclonality and specific ALL markers if present in the patient.In experiment 2, a series of 22 immunodeficient female mice was injected with specific doses of the leukemia cell line RCH-ACV (103 - 5 × 106, n = 4/group) to assess the engraftment competence of the SCID model. MAIN RESULTS AND THE ROLE OF CHANCE: ALL metastatic cells were detected, by PCR, in five DXM-treated and one control human ovarian tissue graft as well as in a control mouse liver, although malignant cell infiltration was not detected by histology in any sample after 6 months. In total, minimal residual disease was present in three DXM-treated and three control mice.RCH-ACV cells were detected in liver and spleen samples after the injection of as little as 103 cells, although only animals receiving 5 × 106 cells developed clinical signs of disease and metastases. LIMITATIONS, REASONS FOR CAUTION: This is an experimental study where the malignant potential of leukemic cells contained in human ovarian tissues has been assessed in immunodeficient mice. WIDER IMPLICATIONS OF THE FINDINGS: These results indicate that DXM incubation prior to retransplantation of ovarian tissue does not prevent reintroduction of leukemic cells. Therefore, caution should be taken in retransplanting ovarian tissue from patients with leukemia until safer systems are developed, as leukemic cells present in ovarian grafts were able to survive, proliferate and migrate after cryopreservation and xenograft. STUDY FUNDING/COMPETING INTEREST(S): Funded by the Regional Valencian Ministry of Education (PROMETEO/2018/137) and by the Spanish Ministry of Economy and Competitiveness (PI16/FIS PI16/01664 and PTQ-16-08222 for S.H. participation). There are no competing interests.

Assessment of reflectance confocal microscopy for non-invasive selection of optimal ovarian cortex fragments for autotransplantation.

RESEARCH QUESTION: Can reflectance confocal microscopy (RCM) be used to determine follicle density in human ovarian cortex fragments that are intended for fertility restoration? DESIGN: RCM was used on living cortex tissue fragments derived from five bovine ovaries and 13 human ovaries. All tissue fragments were cryopreserved and thawed before RCM analysis. Follicle numbers and distribution were determined by RCM and histology. Before and after RCM, general tissue viability and follicle integrity were assessed by a glucose uptake assay and neutral red staining, respectively. RESULTS: RCM can detect all stages of follicle development in living ovarian tissue to a maximum depth of 250 µm. In bovine tissue, all follicles were located within this 0-250 µm range. In human ovarian tissue, follicles were also present below the 250 µm RCM threshold, implying that only a percentage of the total number of follicles could be detected with RCM. The percentage of follicles detected by RCM appeared to be age dependent. The RCM procedure did not affect the glucose uptake by the tissue, whereas neutral red staining indicated a high level of follicle survival. CONCLUSION: In this proof of concept study, we have shown that RCM is a promising technique to determine the density of follicles ex vivo in living human ovarian cortex fragments, apparently without compromising the vitality of the tissue. Safety studies and further optimization of the RCM technique with a focus on increasing the penetration depth are required before clinical use of RCM.

Fertility Preservation in Breast Cancer.

As more young women survive breast cancer, fertility preservation (FP) is an important component of care. This review highlights the importance of early pretreatment referral, reviews the risks of infertility associated with breast cancer treatment, and defines existing and emerging techniques for FP. The techniques reviewed include ovarian suppression, embryo cryopreservation, oocyte cryopreservation, and ovarian tissue cryopreservation and transplantation. The barriers women face, such as not being appropriately referred and the costs of treatment, also are addressed. Multidisciplinary, patient-centered care is essential to discussing FP with patients with breast cancer and ensuring appropriate care that includes quality of life in survivorship.

Accuracy and safety verification of ovarian reserve assessment technique for ovarian tissue transplantation using optical coherence tomography in mice ovary.

Except for histological study, there are currently no suitable techniques available for the detection and identification of primordial follicles in ovary of primary ovarian insufficiency patients who have undetectable AMH levels. Also, the ability to locate and quantify follicles on ovarian cortex strips, without fixation, is valuable for patients who could undergo subsequent successful ovarian tissue transplantation. Although optical coherence tomography (OCT) is a well-established high resolution imaging technique without fixation commonly applied in biomedicine, few reports are available on ovarian tissue imaging. In present study, we established standard OCT follicle images at each developmental stage, including the primordial follicle, and demonstrated the efficacy of OCT to estimate IVF outcome in transplanted mice ovary like ovarian reserve tests. Unfortunately, the current commercial OCT could not be used to accurate follicle count the number of follicles for whole ovary, because the maximum depth of examination was 100 µm. And we demonstrated the safety of OCT examination, it did not affect IVF outcome and birth defect rate, and reproductive ability. Although there is room for improvement, these findings will be first step to bring OCT examination a step closer to clinical application for measuring true ovarian reserve and localizing follicles.

Assessment of the effect of different vitrification solutions on human ovarian tissue after short-term xenotransplantation onto the chick embryo chorioallantoic membrane.

Chemotherapy can lead to the loss of fertility and premature ovarian failure in young women who suffer from malignant diseases. Freezing ovarian tissue by vitrification allows for the preservation of a large number of follicles prior to treatment, yet no established protocols have been optimized with respect to the vitrification solution. The aim of the present study was to evaluate the early stage of human ovarian tissue xenotransplantated onto the chick embryo chorioallantoic membrane after vitrification, and to determine the effect of different vitrification solutions on ovarian tissue quality-as defined by morphology and viability of follicles, neovascularization, cell proliferation, and apoptosis. Each vitrification protocol had a different impact on ovarian tissue at the early transplantation stage; one process using the lowest concentrations of ethylene glycol and dimethylsulfoxide, plus sucrose, demonstrated a moderate advantage compared to the other protocols. We also demonstrated that the chorioallantoic membrane model can be a useful alternative for short-term xenotransplantation studies of angiogenesis into human ovarian cortical tissue.

New methods to improve the safety assessment of cryopreserved ovarian tissue for fertility preservation in breast cancer patients.

OBJECTIVE: To develop a novel molecular panel of markers to detect breast cancer (BC) disseminated malignant cells in ovarian tissue, and to improve the safety of ovarian tissue transplantation. DESIGN: Experimental study. SETTING: University hospital. PATIENT(S): Ten ovarian biopsies from healthy patients, 13 biopsies with diagnosed BC metastasis, and 4 biopsies from primary BC tumor for designing a diagnostic panel of BC cell contamination; 60 ovarian biopsies from BC patients undergoing fertility preservation for validating the panel. ANIMAL(S): Female nude mice. INTERVENTION(S): A novel panel for BC malignant cell detection by reverse-transcription polymerase chain reaction (RT-PCR), inmmunohistochemical analysis, in vitro invasion assay and xenotransplantation assayed in ovarian tissue from BC patients. MAIN OUTCOME MEASURE(S): Expression of GCDFP15, MGB1, SBEM, MUC1, WT-1, and NY-BR-01, selected as markers, assessed by quantitative RT-PCR in samples with confirmed BC metastasis. The most sensitive markers were confirmed by immunohistochemistry, and tested in vitro and in vivo. RESULT(S): GCDFP15, MGB1, and SBEM were the most sensitive and specific markers to detect BC metastatic cells when at least one was expressed by quantitative RT-PCR. The panel was validated in 60 patients and confirmed in an in vitro invasion assay, where no invasive cells were observed. Samples negative for BC cells cannot develop disease when xenografted. CONCLUSION(S): GCDFP15, MGB1, and SBEM were the most sensitive molecules to create a diagnostic panel for BC malignant cell contamination, which may make ovarian tissue cryopreservation and transplantation a safe technique for fertility preservation in BC patients.

Assessment of vitrification outcome by xenotransplantation of ovarian cortex pieces in γ-irradiated mice: morphological and molecular analyses of apoptosis.

PURPOSE: The aim of this study was the investigation of caspase-3/7 activity and apoptosis related gene expression after vitrification and xenotransplantation of human ovarian fragments. METHODS: Ovarian specimens were obtained from normal female-to-male transsexual women during laparoscopic surgery and cut into small pieces and were considered as vitrified and non-vitrified groups. The morphological study, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, caspase-3/7 activity and apoptosis related gene expression analysis were done in both non-vitrified and vitrified groups in two steps (before transplantation of ovarian tissues and 30 days after transplantation). RESULT(S): In spite of high rate of normal follicles in both non-transplanted tissues these rates were significantly decreased in vitrified and non-vitrified grafted tissues, moreover grafted-vitrified tissue showed significantly less normal follicles than grafted-non-vitrified group (P < 0.05). The expression of some pro and anti-apoptotic genes in vitrified-warmed tissues were not changed compared to non-vitrified ones but the expression of Fas and caspase8 was increased and the expression of BRIC5 was decreased in this group (P < 0.05). In transplanted vitrified group the Bcl2, FasL and BRIC5 gene expression was high and caspase8 was low (P < 0.05). The expression of all genes in both grafted groups was more than non-grafted tissues except for caspase8 (P < 0.05). The TUNEL positive signals and caspase-3/7 activity were increased in both grafted groups compared to non-grafted groups and this enzyme activity in grafted-vitrified group was more than grafted-non-vitrified group (P < 0.05). CONCLUSION(S): This study provides the first evidence on the significant effect of vitrification on follicular apoptosis of grafted human ovarian tissue at mRNA level. The signs of follicular survival or degeneration detected by morphological assessment and caspase-3/7 activity were closely correlated to the changes in expression of apoptosis-related genes.

Autologous and allogeneic ovarian orthotopic transplantation: morphologic, endocrinologic and natural pregnancy assessment.

PURPOSE: To assess pregnancy of rabbits submitted to bilateral ovariectomy and orthotopic allogeneic or autologous intact and sliced ovarian transplantation without a vascular pedicle and to determine the morphofunctional aspects of the transplanted ovaries. METHODS: Fifty-six female rabbits were studied. The ovaries were removed and orthotopically transplanted or replaced without vascular anastomoses: Group 1 (n=8), only laparotomy and laparorrhaphy, Group 2A (n=8) intact ovaries were transplanted on both sides, Group 2B (n=8) both ovaries were sliced and orthotopically transplanted, Group 2C (n=8), an intact ovary was transplanted on one side and a sliced ovary on the other side. In Group 3 the ovaries were reimplanted according to the procedure and subgroups described for Group 2. Three months later, the animals were paired with males for copulation. Estradiol, progesterone, FSH and LH hormone levels were assessed. A histologic study was carried out, and the number of pregnancies and litters were also determined. Chi-square test compared the number of pregnancies and litters. One-way ANOVA and the Tukey-Kramer tests compared the hormonal dosages. RESULTS: Pregnancies occurred in seven (87.5%) rabbits of Group 1, in three rabbits (37.5%) of Groups 2A and 3A, in four rabbits (50%) of groups 2B, 3B and 3C, and in five (62.5%) of group 2C. Normal hormone serum levels and histology confirmed the vitality of all ovaries. CONCLUSION: Orthotopic allogeneic and autologous ovarian transplantation without a vascular pedicle is viable in rabbits, and preserves their hormonal levels and fertile functions.

Autologous and allogeneic ovarian orthotopic transplantation: morphologic, endocrinologic and natural pregnancy assessment

PURPOSE: To assess pregnancy of rabbits submitted to bilateral ovariectomy and orthotopic allogeneic or autologous intact and sliced ovarian transplantation without a vascular pedicle and to determine the morphofunctional aspects of the transplanted ovaries. METHODS: Fifty-six female rabbits were studied. The ovaries were removed and orthotopically transplanted or replaced without vascular anastomoses: Group 1 (n=8), only laparotomy and laparorrhaphy, Group 2A (n=8) intact ovaries were transplanted on both sides, Group 2B (n=8) both ovaries were sliced and orthotopically transplanted, Group 2C (n=8), an intact ovary was transplanted on one side and a sliced ovary on the other side. In Group 3 the ovaries were reimplanted according to the procedure and subgroups described for Group 2. Three months later, the animals were paired with males for copulation. Estradiol, progesterone, FSH and LH hormone levels were assessed. A histologic study was carried out, and the number of pregnancies and litters were also determined. Chi-square test compared the number of pregnancies and litters. One-way ANOVA and the Tukey-Kramer tests compared the hormonal dosages. RESULTS: Pregnancies occurred in seven (87.5%) rabbits of Group 1, in three rabbits (37.5%) of Groups 2A and 3A, in four rabbits (50%) of groups 2B, 3B and 3C, and in five (62.5%) of group 2C. Normal hormone serum levels and histology confirmed the vitality of all ovaries. CONCLUSION: Orthotopic allogeneic and autologous ovarian transplantation without a vascular pedicle is viable in rabbits, and preserves their hormonal levels and fertile functions.

Assessment of long-term function of heterotopic transplants of vitrified ovarian tissue in cynomolgus monkeys.

BACKGROUND: Ovarian tissue cryopreservation by rapid cooling (vitrification) is a convenient fertility preservation option. However, the progress of vitrified ovarian tissue after transplantation is not well understood in primates. METHODS: For tissues from cynomolgus monkeys, we used closed straw vitrification and open cryosupport vitrification in which tissues are immersed directly into liquid nitrogen. Following warming, ovarian cortical pieces were autotransplanted and their function was monitored by computed tomography (CT), hormone assays and oocyte recovery, ICSI and embryo transfers (ETs). RESULTS: Hormone cycles were restored in 6 of 7 animals in a mean of 126 days with no significant difference between the two vitrification regimens. The presence of new blood vessels supplying the grafted ovarian tissue was confirmed by contrast-enhanced CT. Oocyte retrieval from two monkeys after transplantation of the ovarian cortex vitrified by cryosupport vitrification yielded a total of nine oocytes of which six fertilized after ICSI, but ETs did not lead to any pregnancies. CONCLUSIONS: This work shows that CT can give insight into ovarian function after heterotopic transplantation, and that heterotopic autografts of vitrified ovarian cortex can give rise to long-term ovarian function and embryos in a primate model. It remains to be established how outcomes following rapid vitrification compared with outcomes following conventional slow cooling procedures.

Assessment of long term endocrine function after transplantation of frozen-thawed human ovarian tissue to the heterotopic site: 10 year longitudinal follow-up study.

PURPOSE: To assess the longevity of ovarian grafts in five cancer patients who underwent heterotopic autotransplantation of frozen-thawed ovarian tissue. METHOD(S): Five cancer survivors underwent heterotopic ovarian transplantation between 2001 and 2011. Stored ovarian tissue (for 1-10 years) was rapidly thawed and transplanted into the space between the rectus sheath and the rectus muscle (8-20 cortical sections per patient). Endocrine function was assessed by monthly blood tests (FSH, LH, E2, progesterone and testosterone) and ultrasound after transplantation. The monitoring was continued until the cessation of endocrine function by consecutive blood tests (E2 < 20 pg/ml; FSH ≥ 35 IU/L). RESULT(S): Endocrine function was restored in all patients between 12-20 weeks after transplantation. Four patients required the second transplantation one to two years after the first transplantation. The duration of endocrine function after the second transplantation was much longer (9 months-84 months). The longest duration of endocrine function was seen in a woman who underwent ovarian transplantation in 2003 and 2004 after radiotherapy for cervical cancer. Even more than seven years after transplantation, endocrine function has not ceased (FSH 9.5, E2 108, on July 1, 2011). Of note, this patient underwent three IVF cycles in 2004 which resulted in four embryos. CONCLUSION(S): Long-term endocrine function lasting for seven years can be established with heterotopic transplantation of cryobanked human ovarian tissue. Re-establishment of long-term endocrine function after ovarian transplantation will benefit young cancer survivors with premature ovarian failure.

Fertility preservation for social indications: a cost-based decision analysis.

OBJECTIVE: Age-related infertility remains a problem that assisted reproductive techniques (ART) have limited ability to overcome. Correspondingly, because an increasing number of women are choosing to delay childbearing, fertility preservation strategies, initially intended for patients undergoing gonadotoxic therapies, are being applied to this group of healthy women. Studies supporting the effectiveness of this practice are lacking. DESIGN: Decision analytic techniques. SETTING: We compared the cost-effectiveness of three strategies for women planning delayed childbearing until age 40: oocyte cryopreservation at age 25, ovarian tissue cryopreservation (OTC) at age 25, and no assisted reproduction until spontaneous conception had been attempted. PATIENT(S): Not applicable. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Cost-effectiveness, which was defined as the cost per live birth. RESULT(S): In this analysis, the strategy of foregoing fertility preservation at age 25 and then choosing ART only after not spontaneously conceiving at age 40 was the most cost-effective option. OTC was dominated by the other strategies. Sensitivity analyses demonstrated the robustness of the model; no analysis existed in which OTC was not dominated by oocyte cryopreservation. Increasing the cost of an IVF cycle beyond $22,000 was the only situation in which oocyte cryopreservation was the most preferred strategy. CONCLUSION(S): Neither oocyte cryopreservation nor OTC appear to be cost-effective under current circumstances for otherwise healthy women planning delayed childbearing. This analysis should give pause to the current practice of offering fertility preservation based only on the desire for delayed childbearing.

Assessment of canine ovaries autografted to various body sites.

The influence of graft site on the survival of canine follicles and oocytes after autografting was investigated. Hemi-ovaries were autografted to three locations (quadriceps femoris muscle fascia, kidney capsule, and gastrosplenic ligament), and grafted ovaries were recovered (under anesthesia) 28 to 31 d after transplantation. The grafted hemi-ovaries were bisected: one-quarter ovary was used for histological assessment and another quarter for evaluation of oocyte viability. As controls, the remaining fresh hemi-ovaries were used to assess the viability of follicles and oocytes in non-transplanted ovaries. Most follicles in the histological sections of the grafts were classified as primordial or primary follicles. Antral follicles were not observed in the grafts, irrespective of the graft site. The percentages of viable follicles in the sections from control ovaries, and the ovaries grafted to the kidney capsule, the quadriceps femoris muscle fascia, and the gastrosplenic ligament were 17.4, 22.9, 18.3, and 32.4%, respectively. A total of 12 oocytes was recovered from the 15 hemi-ovaries grafted in five bitches, of which five (41.7%) oocytes from the ovaries grafted to the quadriceps femoris muscle fascia and the kidney capsule were cultured for assessment of meiotic competence. Three oocytes were viable but remained in the germinal vesicle stage after 72 h of maturation culture. The quadriceps femoris muscle fascia might be useful for grafting like the kidney capsule, but improvement of follicle survival and meiotic competence of oocytes in the grafts is necessary.

Autotransplantation of cryopreserved ovarian tissue: a procedure with promise, risks, and a need for a registry.

More and more women are being referred for discussion of fertility preservation options. The ability to efficiently and safely preserve the reproductive potential of thousands of young female cancer patients looks possible, but many issues are not yet resolved. Where is the best place for autotransplantation of ovarian tissue? How long would this tissue be expected to function? What is the chance of hormone production and/or pregnancy from this tissue? What is the risk of recurrent cancer from autotransplantation of ovarian tissue? An accurate assessment of the risks and likelihood of success is critical for appropriately counseling these women.

Cryopreservation and orthotopic transplantation of rat ovaries.

The number of rat strains increased considerably in the last decade and will increase continuously during the next years. This requires enough space for maintaining vital strains and techniques for cryobanking, which can be applied not only in specialised rat resource centres but also in regular animal houses. Here we describe an easy and fast method for the cryopreservation and transplantation of frozen-thawed ovaries of the rat. With dimethyl sulfoxide as cryoprotectant rat ovaries can be stored at -196 degrees C for unlimited time. For revitalisation thawed ovaries have to be orthotopically transplanted into appropriate ovarectomised recipients. Reestablishment of the reproductive cycle in the recipients can be confirmed by vaginal cytology shortly after transplantation. The recipients are able to produce 2-3 litters after mating with males of an appropriate strain. Cyropreservation of ovaries thus can be considered a reliable method to preserve scientifically and economically important stocks and strains of rats that are currently not required.

Assessment of vascular endothelial growth factor expression and apoptosis in the ovarian graft: can exogenous gonadotropin promote angiogenesis after ovarian transplantation?

OBJECTIVE: To evaluate the effects of gonadotropin on angiogenesis by assessing vascular endothelial growth factor (VEGF) expression in rat ovaries transplanted after freezing and thawing. DESIGN: In vitro laboratory experiments. SETTING: Academic research institute. ANIMAL(S): Sixty immature female rats. INTERVENTION(S): Frozen-thawed ovaries were autotransplanted into the SC tissue of 60 rats (ages between 21 and 28 days). After transplantation, either pregnant mare's serum gonadotropin (PMSG) or saline was administered. The grafted ovaries were collected 2, 7, and 30 days after transplantation for evaluation. MAIN OUTCOME MEASURE(S): Assessment of the morphology and number of follicles, evaluation of apoptosis, and analysis of VEGF expression in the grafted ovaries. RESULT(S): Most follicles in the grafts were apoptotic on day 2 but recovered by day 7. The proportion of antral follicles and corpora lutea in the graft correlated with the duration after transplantation. A significant increase in the expression of VEGF188 mRNA was noticed in the grafted ovaries on day 2. Moreover, the mRNA expression in the PMSG group was higher than that in the control group. The increased VEGF protein production in the graft was confirmed by Western blot analysis. CONCLUSION(S): In ovariectomized animals, gonadotropin treatment may not provide any added benefits for folliculogenesis and angiogenesis. Nevertheless, a significant increase in the VEGF188 isoform in the gonadotropin-treated group may suggest the positive effect of exogenous gonadotropin therapy in the early stages of angiogenesis.