Effects of different subcutaneous sites on heterotopic autotransplantation of canine ovarian tissue.

Ovarian tissue transplantation makes it possible to restore fertility; however, the success of this technique depends on the transplant region used. Therefore, this study aimed to evaluate the effect of two subcutaneous regions on canine ovarian transplantation, pinna (Pi) and neck (Ne), for 7 and 15 days. Ovaries collected by ovariosalpingohysterectomy were fragmented using a punch device. Fresh fragments were fixed, and the others were immediately grafted onto the animal itself in the Pi and Ne regions for 7 and 15 days. Recovered fragments were evaluated for histology (morphology, development and stromal density), picrosirius (collagen fibers), and immunohistochemistry (fibrosis and cell proliferation). The results showed that follicular normality rates were lower in Pi-7 (78%) vs. control (90%) and Pi-15 (86%), similar in Ne-7 (92%) and superior in Ne-15 (97%) compared to the control, with the effect of the region Ne (94%) superior (P < 0.05) to Pi (82%). Stromal density reduced in both regions vs. control but was similar within 15 days. Fragments from both regions showed higher fibronectin labeling and deposition of type I and lower type III collagen fibers (P < 0.05) vs. control. Proliferation rates in Ne-7 were higher (P < 0.05) than in control, and Pi-15 was higher (P < 0.05) than Ne-15. In conclusion, the pinna may be a region with greater potential than the neck after a 15-day autotransplantation of canine ovarian tissue.

Fertility preservation: improved neovascularization and follicle viability in cryopreserved bovine ovarian cortex transplants by remaining medulla tissue.

OBJECTIVE: To investigate the advantages of cryopreserved medulla-containing ovarian cortex grafts with those of commonly used sole cortex grafts for fertility preservation by analyzing tissue quality, neovascularization processes, and the number of vital follicles. DESIGN: Experimental setting of cryopreserved bovine ovarian cortex tissue grafts with or without medulla tissue. SETTING: Laboratory animal research at Ulm University, Ulm, Germany. ANIMALS: Bovine ovaries and fertilized chicken eggs. INTERVENTION(S): Experimental setting of bovine ovarian tissue samples grafted on the chicken chorioallantoic membrane (CAM) after cryopreservation and thawing to examine histologic tissue integrity, apoptosis and proliferation immunohistochemically, blood vessel counts and determine the presence of neutral red-stained vital follicles. MAIN OUTCOME MEASURE(S): We used hematoxylin and eosin staining to visualize tissue structures, immunohistochemistry with anti-caspase 3 to detect apoptosis, anti-Ki67 to examine proliferation, blood vessel count on the chicken CAM to investigate neovascularization processes, and neutral red staining to evaluate vital follicles. RESULT(S): We demonstrated that in all analyzed tissue samples, after cryopreservation, thawing, and grafting on the chicken CAM, there was excellent tissue integrity and quality, as shown by extremely rare apoptosis processes analyzed using immunohistochemical caspase 3 staining (sole cortex, 0.54%; thin medulla-containing cortex, 0.43%; thick medulla-containing cortex, 0.13%; and sole medulla, 2.82%). Moreover, we detected increased neovascularization in the vicinity of medulla and medulla-containing grafts (small blood vessels: cortex 8.7, thin medulla-containing cortex 9.9, thick medulla-containing cortex 9.7, and medulla 9.8; very small blood vessels: cortex 7.0, thin medulla-containing cortex 13.0, thick medulla-containing cortex 12.0, and medulla 15.0), with higher Ki67-detected proliferation (cortex, 17.58%; thin medulla-containing cortex, 20.28%; thick medulla-containing cortex, 20.56%; and medulla, 29.9%). Additionally, we identified an increased number of vital follicles in medulla-containing cortex grafts compared with the number of vital follicles in sole cortex tissue (cortex, 256.1; thin medulla-containing cortex, 338.2; thick medulla-containing cortex, 346.6; and medulla, 8.1). CONCLUSION(S): In this experimental setting, bovine medulla-containing cortex tissue had excellent tissue structure and quality after cryopreservation and thawing and increased neovascularization and an augmented vital follicle count after grafting than the commonly used sole cortex tissue. Therefore, we suggest reconsidering the current cryopreservation and grafting processes in humans for fertility preservation by favoring retain medulla tissue at the ovarian cortex.

Does it make sense to refreeze ovarian tissue after unexpected occurrence of endometriosis when transplanting the tissue?

BACKGROUND: Ovarian insufficiency is a major concern for long-term cancer survivors. Ovarian tissue cryopreservation for fertility preservation is an emerging technique that has proven successful over the past decade through transplantation of frozen-thawed ovarian tissue. Compared to other established techniques, such as oocyte freezing, ovarian tissue cryopreservation preserves actual organ function and thus the production of sex hormones. Endometriosis in perimenopausal women is rare, however it can be surprising diagnosis in the planned transplantation of cryopreserved ovarian tissue and the already thawed tissue may not be transplanted, so that it has to be refrozen. RESULTS: Ovarian function returned in the patient two months after transplantation, as shown by estrogen production. Ten months after the ovarian tissue transplantation mild stimulation with FSH was initiated in accordance with a low-dose protocol. When ultrasonography revealed a follicle 17 mm in size in the ovarian graft, hCG was added and after follicular puncture one oocyte was obtained. The oocyte could be fertilized by IVF and transferred to the uterus. On day 14 after embryo-transfer, a positive hCG-Level was detected and after an uncomplicated pregnancy a healthy child was delivered. CONCLUSIONS: We report the first pregnancy and live birth achieved using transplantation of thawed and refrozen ovarian tissue in a woman treated by chemotherapy and subsequent endometriosis surgery. Refreezing of cryopreserved ovarian tissue is not a hindrance to successful transplantation of ovarian tissue. Against the background of increasing numbers of candidates for transplantation of ovarian tissue is expected that the combination chemotherapy followed by endometriosis will increase.

Revascularization of human ovarian grafts is equally efficient from both sides of the cortex tissue.

RESEARCH QUESTION: Does revascularization of human ovarian grafts in a mouse model occur with equal efficiency from both sides of the cortex tissue? DESIGN: Twenty-four frozen-thawed ovarian cortex pieces from 12 women were transplanted to immunodeficient mice, for 8 days to analyse graft revascularization using immunohistochemical detection of murine CD31, or for 8 weeks to evaluate follicle density (follicles/mm3). The CD31-positive vessel area and density were quantified using a custom-designed application. Three regions of interest (ROI) were defined in each tissue section: the cortical side, the centre and the medullary side. Vessels were subdivided into three categories according to size: microvessels (<300 µm2), small vessels (300-1000 µm2) and large vessels (>1000-3000 µm2). RESULTS: No significant difference in the mean percentage of the CD31-positive vessel area was found between the three ROI (cortical side: 3.9% ± 0.2%; centre: 3.5% ± 0.2%; medullary side: 4.0% ± 0.3%; P = 0.17), but a significantly lower density of vessels was found in the centre of the human ovarian grafts compared with the cortical and medullary sides (cortical side: 323 ± 14 vessels/mm2; centre: 240 ± 12 vessels/mm2; medullary side: 301 ± 18 vessels/mm2; P < 0.001). Microvessels comprised 89-91% of all vessels in the three ROI. Follicle density in ungrafted cortex pieces was 51.8 ± 17.3 and 14.7 ± 3.7 follicles/mm3 after 8 weeks of xenografting, resulting in a follicle survival rate of 28%. CONCLUSIONS: Host revascularization was established equally efficiently from both sides of transplanted human ovarian cortex, suggesting that transplantation techniques ensuring revascularization from both sides of the ovarian graft could potentially facilitate faster graft revascularization.

Role of Stem Cells in the Ovarian Tissue Cryopreservation and Transplantation for Fertility Preservation.

Although the cancer survival rate has increased, cancer treatments, including chemotherapy and radiotherapy, can cause ovarian failure and infertility in women of reproductive age. Preserving fertility throughout cancer treatment is critical for maintaining quality of life. Fertility experts should propose individualized fertility preservation methods based on the patient's marital status, pubertal status, partner status, and the urgency of treatment. Widely practiced fertility preservation methods, including ovarian transposition and embryo and oocyte cryopreservation, are inappropriate for prepubertal girls or those needing urgent initiation of cancer treatment. Ovarian tissue cryopreservation and transplantation, an emerging new technology, may be a solution for these cancer patients. The use of stem cells in ovarian tissue cryopreservation and transplantation increases oxygenation, angiogenesis, and follicle survival rates. This review discusses the recent advances in ovarian tissue cryopreservation and transplantation with special focus on the use of stem cells to improve fertilization techniques.

Taurine improves follicular survival and function of mice ovarian grafts through increasing CD31 and GDF9 expression and reducing oxidative stress and apoptosis.

Ischemia-reperfusion (IR) injury is a major limitation of ovarian transplantation which threatens the follicular and graft survival. Taurine as a potent anti-oxidant, anti-apoptotic and anti-inflammatory agent, can prevent graft damages due to IR. We aimed to investigate the effect of taurine on the follicular survival and function of autotransplanted mouse ovaries. Female mice (4-5 weeks old) were divided into: control, autograft and autograft + taurine (200 mg/kg/day). The level of CD31 expression was evaluated two days (48 h) post transplantation. In addition, on day 7 post transplantation the serum levels of malondialdehyde (MDA) and the total antioxidant capacity (TAC) were assessed. Also, 28 days post transplantation; ovaries were studied stereologically and the percentage of apoptotic follicles, level of GDF9 expression and the serum concentrations of progesterone and estradiol were measured. Data were analyzed using one-way ANOVA and Tukey's test and the means were considered significantly different at P < 0.05. The total volume of the ovary (P < 0.01), volume of the cortex (P < 0.01) and medulla (P < 0.04), total number of different types of follicles, expression of GDF9 and CD31 and also the levels of progesterone, estradiol and TAC increased significantly in the autograft + taurine group compared to the autograft group (P < 0.001). The MDA level and apoptosis rate decreased significantly in the autograft + taurine group compared to the autograft group (P < 0.001). Taurine could significantly improve follicular survival and the function of grafted ovaries by accelerating the angiogenesis and reducing oxidative stress and apoptosis.

Alpha Lipoic Acid Supplementation Improves Ovarian Tissue Vitrification Outcome: An Alternative to Preserve the Ovarian Function of Morada Nova Ewe.

This study evaluated the effect of adding alpha lipoic acid (ALA) to the vitrification solution of sheep ovarian tissue on 7 days of in vitro culture or 15 days of xenotransplantion. ALA was used at two different concentrations (100 µM: ALA100 and 150 µM: ALA150). Ovarian tissue was evaluated by classical histology (follicular morphology, development, and stromal cell density); immunohistochemistry for forkhead box O3a (FOXO3a); Ki67 (cell proliferation); cluster of differentiation 31 (CD31); and alpha smooth muscle actin (α-SMA). Reactive oxygen species (ROS) levels in ovarian tissue, as well as malondialdehyde (MDA) and nitrite levels in the culture medium, were assessed. Similar percentage of morphologically normal follicles was found in the vitrified ovarian tissue in the presence of ALA100 or ALA150 after in vitro culture or xenotransplantation. Follicular development from all treatments was higher (P < 0.05) than the control group. Moreover, an activation of primordial follicles was observed by FOXO3a. Stromal cell density and immunostaining for Ki67 and CD31 were significantly higher (P < 0.05) in ALA150 vitrified tissue. No difference (P > 0.05) was found in α-SMA between ALA concentrations after in vitro culture or xenograft. ROS levels in the ovarian tissue were similar (P > 0.05) in all treatments, as well as MDA and nitrite levels after 7 days of culture. We concluded that the addition of ALA 150 is able to better preserve the stromal cell density favoring granulosa cell proliferation and neovascularization.

Disease-inducing potential of two leukemic cell lines in a xenografting model.

PURPOSE: Cryopreserved ovarian tissue transplant restores ovarian function in young cancer patients after gonadotoxic treatment. However, leukemia is associated with increased risk of malignant cell transmission. We aimed to assess the tumor-inducing potential of two different leukemic cell lines when xenografted to immunodeficient mice. METHODS: Fifty-four female immunodeficient mice were grafted with either 100, 200, 500, 1000, and 10,000 chronic myeloid leukemia in blast crisis (BV-173) cells or relapsed acute lymphoblastic leukemia (RCH-ACV) cells, embedded inside a fibrin scaffold along with 50,000 human ovarian stromal cells. Two mice per cell line received the fibrin matrix without leukemic cells as negative controls. Clinical signs of disease were monitored for 20 weeks. Grafts, liver tissue, and masses were collected for macroscopic analysis and gene expression of BCR-ABL1 and E2A-PBX fusion transcripts present in BV-173 and RCH-ACV respectively. RESULTS: BV-173 cells: Mice grafted with 100, 200, or 500 cells showed no sign of disease after and were negative for BCR-ABL1 expression. Three of the 5 animals grafted with 1000 cells and all mice with 10,000 cells developed disease and showed BCR-ABL1-positive expression. RCH-ACV cells: Two out of 4 mice grafted with 100 cells developed disease and were E2A-PBX1-positive. All the animals grafted with higher cell doses showed signs of disease and all but one were E2A-PBX1-positive. CONCLUSION: The present work proves that the disease-inducing potential of BV-173 and RCH-ACV leukemic cells xenografted to SCID mouse peritoneum differs between cell lines, depending on cell number, type, status, and cytogenetic disease profile when ovarian tissue is harvested.

Efficacy of assisted reproductive technology after ovarian tissue transplantation in a cohort of 11 patients with or without associated infertility factors.

PURPOSE: IVF treatment in women with grafted frozen-thawed ovarian tissue is associated with poor reproductive outcomes. The aim of this study was to evaluate the efficacy of ovarian tissue transplantation (OTT) followed by assisted reproductive technology (ART) in women with or without associated infertility factors. METHODS: This is a prospective cohort study with retrospective data collection including eleven women, four of whom having an infertility factor (IF), who had undergone OTT in one university center between 2005 and 2017, followed by ART in six in vitro fertilization (IVF) centers. RESULTS: In total, 25 of the 85 cycles initiated (29%) were canceled, resulting in 60 oocyte retrievals. Ninety-five oocytes were retrieved: 36 were abnormal or immature, 29/39 fertilized (74%) after ICSI and 13/20 (65%) after IVF. Thirty-five embryos were transferred in seven patients (5/7 patients without IF and 2/4 patients with IF). After ART, one patient with IF experienced two pregnancies, one resulting in a live birth. For all patients, pregnancy rates and live birth rates were 7.4% and 3.7% per embryo transfer, respectively. Nine pregnancies and four live births occurred after spontaneous conception in five patients without IF, none in the infertility group. CONCLUSION: This study confirms that IVF treatment in women with grafted frozen-thawed ovarian tissue is associated with poor outcomes. However, the chances of natural conception are high in women without IF. Patients with IF, without the possibility of spontaneous pregnancy, should be informed of poor reproductive outcomes after OTT followed by ART. TRIAL REGISTRATION NUMBER: NCT02184806.

Adipose tissue-derived stem cells protect the primordial follicle pool from both direct follicle death and abnormal activation after ovarian tissue transplantation.

PURPOSE: To investigate whether adipose tissue-derived stem cells (ASCs) protect the primordial follicle pool, not only by decreasing direct follicle loss but also by modulating follicle activation pathways. METHODS: Twenty nude mice were grafted with frozen-thawed human ovarian tissue from 5 patients. Ten mice underwent standard ovarian tissue transplantation (OT group), while the remaining ten were transplanted with ASCs and ovarian tissue (2-step/ASCs+OT group). Ovarian grafts were retrieved on days 3 (n = 5) and 10 (n = 5). Analyses included histology (follicle count and classification), immunohistochemistry (c-caspase-3 for apoptosis and LC3B for autophagy), and immunofluorescence (FOXO1 for PI3K/Akt activation and YAP for Hippo pathway disruption). Subcellular localization was determined in primordial follicles on high-resolution images using structured illumination microscopy. RESULTS: The ASCs+OT group showed significantly higher follicle density than the OT group (p = 0.01). Significantly increased follicle atresia (p < 0.001) and apoptosis (p = 0.001) were observed only in the OT group. In primordial follicles, there was a significant shift in FOXO1 to a cytoplasmic localization in the OT group on days 3 (p = 0.01) and 10 (p = 0.03), indicating follicle activation, while the ASCs+OT group and non-grafted controls maintained nuclear localization, indicating quiescence. Hippo pathway disruption was encountered in primordial follicles irrespective of transplantation, with nuclear YAP localized in their granulosa cells. CONCLUSION: We demonstrate that ASCs exert positive effects on the ovarian reserve, not only by protecting primordial follicles from direct death but also by maintaining their quiescence through modulation of the PI3K/Akt pathway.

Heterotopic autotransplantation of ovarian tissue in a large animal model: Effects of cooling and VEGF.

Heterotopic and orthotopic ovarian tissue autotransplantation techniques, currently used in humans, will become promising alternative methods for fertility preservation in domestic and wild animals. Thus, this study describes for the first time the efficiency of a heterotopic ovarian tissue autotransplantation technique in a large livestock species (i.e., horses) after ovarian fragments were exposed or not to a cooling process (4°C/24 h) and/or VEGF before grafting. Ovarian fragments were collected in vivo via an ultrasound-guided biopsy pick-up method and surgically autografted in a subcutaneous site in both sides of the neck in each mare. The blood flow perfusion at the transplantation site was monitored at days 2, 4, 6, and 7 post-grafting using color-Doppler ultrasonography. Ovarian grafts were recovered 7 days post-transplantation and subjected to histological analyses. The exposure of the ovarian fragments to VEGF before grafting was not beneficial to the quality of the tissue; however, the cooling process of the fragments reduced the acute hyperemia post-grafting. Cooled grafts compared with non-cooled grafts contained similar values for normal and developing preantral follicles, vessel density, and stromal cell apoptosis; lower collagen type III fibers and follicular density; and higher stromal cell density, AgNOR, and collagen type I fibers. In conclusion, VEGF exposure before autotransplantation did not improve the quality of grafted tissues. However, cooling ovarian tissue for at least 24 h before grafting can be beneficial because satisfactory rates of follicle survival and development, stromal cell survival and proliferation, as well as vessel density, were obtained.

Early evaluation of survival of the transplanted ovaries through ultrasound molecular imaging via targeted nanobubbles.

Ovarian survival after transplantation is key to determining the success and efficacy of ovarian tissue cryopreservation and transplantation (OTCP). However, non-invasive monitoring of ovarian survival in the early stages of ovarian transplantation remains a great challenge. Anti-Müllerian hormone (AMH) is a survival factor that can promote the growth of follicles and has been recognized as an ovarian tissue-specific marker. In this study, we developed AMH-targeted nanobubbles (NBAMH) by integrating an AMH antibody onto the surface of NBs. The resulting NBAMH exhibited a high affinity for ovarian granulosa cells in vitro and a significantly enhanced ultrasound signal in transplanted ovaries relative to the non-targeted NBs. Notably, the difference in enhanced ultrasonic signals became more significant with the increase in time after transplantation from 3 to 10 days, indicating a gradually enhanced AMH expression along with the increase in transplant time. These results were further confirmed by immunohistochemical staining and western immunoblotting analyses. In conclusion, our study offers a promising non-invasive tool to monitor ovarian survival in the early stages following transplantation.

Follicle populations and vascularization in ovarian tissue of pediatric patients before and after long-term grafting.

OBJECTIVE: To characterize ovarian tissue from pediatric patients by evaluating development and vascularization in follicle populations and comparing it with adult tissue after xenografting. DESIGN: Prospective experimental study. SETTING: Academic research center. PATIENT(S): Five children (median age 3 years) and seven women (median age 28 years). INTERVENTION(S): Hematoxylin and eosin staining, immunofluorescence, and transmission electron microscopy (TEM) evaluation before and after grafting. MAIN OUTCOME MEASURE(S): Follicle density, morphology, classification, and size, ovarian tissue vascularization, follicle ultrastructure. RESULT(S): Frozen-thawed ovarian tissue was divided into three fragments: nongrafted controls, TEM, and xenografting for 20 weeks. Follicle density was statistically significantly higher in pediatric than adult patients; even though it decreased in both groups after transplantation, it remained higher in pediatric patients. In the pediatric group, quiescent-stage follicles were the majority of the follicle pool before and after grafting, while growing follicles statistically significantly increased in both groups after grafting. Abnormal and atretic follicles were also observed in pediatric tissue and declined with age and after grafting. Pediatric ovarian tissue contained more and larger immature vessels, while mature vessels were larger in adults. The TEM analysis of abnormal pediatric follicles showed loss of shape and vacuolization of the cytoplasm without organelle damage. CONCLUSION(S): Statistically significant differences in follicle density were observed between pediatric and adult patients, but the follicle proportions were similar before and after grafting, with the exception of atretic and abnormal follicles. Pediatric tissue contains more and larger immature vessels than adult tissue, and the posttransplantation revascularization process is accelerated in this group.

Novel extra cellular-like matrices to improve human ovarian grafting.

PURPOSE: To investigate if human ovarian grafting with pure virgin human recombinant collagen type-1 from bioengineered plant lines (CollPlant™) or small intestine submucosa (SIS) yields better implantation results for human ovarian tissue and which method benefits more when combined with the host melatonin treatment and graft incubation with biological glue + vitamin E + vascular endothelial growth factor-A. METHODS: Human ovarian tissue wrapped in CollPlant or SIS was transplanted into immunodeficient mice with/without host/graft treatment. The tissue was assessed by follicle counts (including atretic), for apoptosis evaluation by terminal deoxynucleotidyl transferase assay and for immunohistochemical evaluation of neovascularization by platelet endothelial cell adhesion molecule (PECAM) expression, and for identification of proliferating granulosa cells by Ki67 expression. RESULTS: Human ovarian tissue transplanted with CollPlant or SIS fused with the surrounding tissue and promoted neovascularization. In general, implantation with CollPlant even without additives promoted better results than with SIS: significantly higher number of recovered follicles, significantly fewer atretic follicles, and significantly more granulosa cell proliferation. Moreover, results with CollPlant alone seemed to be at least as good as those after host and graft treatments. CONCLUSIONS: CollPlant is a biomaterial without any potential risks, and grafting ovarian tissue with CollPlant is easy and the procedure may be easily modified, with limited or no foreseeable risks, for auto-transplantation in cancer survivors. Further studies are needed using other novel methods capable of enhancing neovascularization and reducing apoptosis and follicle atresia.

First pregnancy and live birth in Turkey following frozen-thawed ovarian tissue transplantation in a patient with acute lymphoblastic leukemia who underwent cord blood transplantation.

PURPOSE: To report the first live birth after frozen-thawed ovarian transplantation in Turkey and the second case for an acute lymphoblastic leukemia (ALL) survivor in the world. METHODS: A 19-year-old patient underwent ovarian tissue cryopreservation (OTC) before cord blood transplantation in 2010. She was diagnosed as ALL with a bone marrow biopsy revealing 90% blast ALL-L2 type, and karyotype analyses indicated reciprocal translocation at t(9;22)(q34;q11). The patient received the Berlin-Frankfurt-Munster (BFM) protocol, and complete remission was achieved before fertility preservation. Serum AMH level was measured as 1.5 ng/ml, and 12 antral follicles were counted on ultrasound. She was informed about fertility preservation options and decided to proceed with OTC, with her signed consent before cord blood transplantation in April 2011. Ovarian tissue transplantation (OTT) was performed in 2017 when the patient was menopausal with serum FSH levels > 100 IU/ml and estradiol < 20 pg/ml and hematologically in molecular remission. Detailed molecular analysis, standard histology, and immunohistochemistry demonstrated that the thawed tissue is free of malignant cells. RESULTS: Six months following OTT, she had spontaneous menstruation with serum FSH 11 IU/ml and estradiol 53 pg/ml. Two consecutive IVF cycles yielded three top-quality embryos. Following three embryo transfer cycles, one fresh and two frozen, a healthy term live birth was achieved. Frozen-thawed-transplanted tissues were extracted during caesarean delivery upon the patient's request after a total period of 25 months in vivo, and histopathological evaluation revealed that the tissue was free of leukemic infiltration. CONCLUSION: The authors report the first pregnancy and live birth in Turkey and the second live birth in the world following transplantation of frozen-thawed ovarian tissue in a leukemia survivor. As the transplanted tissues were removed during caesarean delivery, histological findings prove the functionality and the malignant-free status of the transplanted tissue during the grafted period.

Peroxisome Proliferator-Activated Receptor Gamma Modulator Promotes Neonatal Mouse Primordial Follicle Activation In Vitro.

Peroxisome proliferator-activated receptor gamma (PPARγ) is known as a regulator of cellular functions, including adipogenesis and immune cell activation. The objectives of this study were to investigate the expression of PPARγ and identify the mechanism of primordial follicle activation via PPARγ modulators in mouse ovaries. We first measured the gene expression of PPARγ and determined its relationship with phosphatase and tensin homolog (PTEN), protein kinase B (AKT1), and forkhead box O3a (FOXO3a) expression in neonatal mouse ovaries. We then incubated neonatal mouse ovaries with PPARγ modulators, including rosiglitazone (a synthetic agonist of PPARγ), GW9662 (a synthetic antagonist of PPARγ), and cyclic phosphatidic acid (cPA, a physiological inhibitor of PPARγ), followed by transplantation into adult ovariectomized mice. After the maturation of the transplanted ovaries, primordial follicle growth activation, follicle growth, and embryonic development were evaluated. Finally, the delivery of live pups after embryo transfer into recipient mice was assessed. While PPARγ was expressed in ovaries from mice of all ages, its levels were significantly increased in ovaries from 20-day-old mice. In GW9662-treated ovaries in vitro, PTEN levels were decreased, AKT was activated, and FOXO3a was excluded from the nuclei of primordial follicles. After 1 month, cPA-pretreated, transplanted ovaries produced the highest numbers of oocytes and polar bodies, exhibited the most advanced embryonic development, and had the greatest blastocyst formation rate compared to the rosiglitazone- and GW9662-pretreated groups. Additionally, the successful delivery of live pups after embryo transfer into the recipient mice transplanted with cPA-pretreated ovaries was confirmed. Our study demonstrates that PPARγ participates in primordial follicle activation and development, possibly mediated in part by the PI3K/AKT signaling pathway. Although more studies are required, adapting these findings for the activation of human primordial follicles may lead to treatments for infertility that originates from poor ovarian reserves.

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.

Investigation on revascularization time and initial damage after transplantation of fresh and cryopreserved ovarian tissue in domestic cats.

The present study evaluated revascularization time of fresh and cryopreserved cat ovarian tissue after transplantation to subcutaneous tissue. Ovaries of five cats were used and eight pieces of ovarian tissue were taken from each pair of ovaries. Immediately after removal, three pieces were transplanted and one fixed for fresh control. The remaining four pieces were cryopreserved and, after thawing, one was fixed for cryopreservation control and three were transplanted. Grafts were recovered on days 2 (D2), 4 (D4) and 6 (D6) post-transplantation. Blood vessels were identified by immunohistochemistry and doppler ultrasound. Immunohistochemistry showed that the percentages of total tissue area occupied by blood vessels were similar (P > 0.05) in fresh and cryopreserved tissues. In both cases, blood vessel area was significantly higher (P < 0.05) on D4 and D6 compared to D0. Ultrasound analysis showed vascularization improvement on the periphery of grafts from D2 to D4 and from D4 to D6, both in fresh and cryopreserved tissue samples. Nonetheless, there was a significant decrease (P < 0.05) in the percentage of morphologically normal follicles (MNF) after transplantation compared to non-transplanted tissue (D0), both for fresh and cryopreserved samples. Moreover, the number of follicles found in samples was considerably smaller after grafting. In conclusion, revascularization of ovarian tissue autotransplanted to subcutaneous tissue in domestic cats occurs within 4 days after transplantation, both for fresh and cryopreserved tissue. However, large follicular loss has been observed in the first days post-transplantation, especially in cryopreserved tissues.

The growth and development conditions in mouse offspring derived from ovarian tissue cryopreservation and orthotopic transplantation.

PURPOSE: To investigate the potential development or metabolic risk in offspring derived from mice with transplanted frozen-thawed ovarian tissue. METHODS: Mice ovaries were intervened by vitrification (group V) and slow-freezing (group S) cryopreservation and orthotopic transplantation. Orthotopic transplantation of fresh ovarian (group F) and natural mating (group C) served as control groups. The fertility restoration and health conditions of generations were assessed by offspring counts, anti-fatigue and motor ability, and organ morphology. The methylation rate and expression level of imprinted genes (IGF2R, H19, SNRPN, and PLAGL1) were used to predict the potential risk of development in transplanted generations. RESULTS: Both the percentage of normal morphological follicles in different developmental periods and the litter size of receipt mice were comparable in all three transplanted groups. There was no significant difference in offspring mice's birth defects, body weight gain, anti-fatigue ability, or exercise capacity among the four groups. The methylation rate of IGF2R, H19, and PLAGL1 showed a significant variation in cryopreservation groups as compared with control groups, as well as a difference in gene expression. The SNRPN appeared to be stable in methylation status. There were no differences in mRNA expression in all groups. CONCLUSIONS: The different ovarian tissue cryopreservation methods did not influence either maternal fertility function or offspring growth. However, these technologies could affect the methylation rate and expression level of some development-related imprinting genes in the offspring, which may lead to some indeterminacy risk.

FERTILITY PRESERVATION: Progress and prospects for developing human immature oocytes in vitro.

Ovarian cryopreservation rapidly developed from basic science to clinical application and can now be used to preserve the fertility of girls and young women at high risk of sterility. Primordial follicles can be cryopreserved in ovarian cortex for long-term storage and subsequently autografted back at an orthotopic or heterotopic site to restore fertility. However, autografting carries a risk of re-introducing cancer cells in patients with blood-born leukaemias or cancers with a high risk of ovarian metastasis. For these women fertility restoration could only be safely achieved in the laboratory by the complete in vitro growth (IVG) and maturation (IVM) of cryopreserved primordial follicles to fertile metaphase II (MII) oocytes. Culture systems to support the development of human oocytes have provided greater insight into the process of human oocyte development as well as having potential applications within the field of fertility preservation. The technology required to culture human follicles is extremely challenging, but significant advances have been made using animal models and translation to human. This review will detail the progress that has been made in developing human in vitro growth systems and consider the steps required to progress this technology towards clinical application.