Idiopathic premature ovarian failure in 63 young women

BACKGROUND: Premature ovarian failure (POF) in adolescents is defined as primary or secondary amenorrhea associated with high follicle-stimulating hormone (FSH) levels. In normal 46,XX patients, its etiology is most often unknown. We have evaluated the clinical, hormonal and ovarian phenotypes in patients with a normal karyotype who were diagnosed with POF before the age of 18. METHODS: Sixty-three patients were included in this retrospective study. RESULTS: The mean patient age was 20.4 years. The patients presented with three clinical patterns: lack of pubertal development (n = 23), primary amenorrhea with interrupted puberty (n = 18), and secondary amenorrhea with normal puberty (n = 22). Ten patients had a familial history of POF and 6 presented with hypothyroidism. The FSH, estradiol and inhibin B levels were not statistically different in the three clinical groups. Fifty percent of the patients presented small ovaries (length <2 cm) at ultrasonography. The presence of follicles was found at histology in only 7 of the 27 patients who underwent an ovarian biopsy. CONCLUSION: 46,XX patients presenting with early POF rarely presented a specific, identifiable disorder. We discuss the clinical management and different diagnosis strategies to improve our current knowledge of this syndrome.     

Homing and Restorative Effects of Bone Marrow-Derived Mesenchymal Stem Cells on Cisplatin Injured Ovaries in Rats

Premature ovarian failure (POF) is a long-term adverse effect of chemotherapy treatment. However, current available treatment regimens are not optimal. Emerging evidence suggests that bone marrow-derived mesenchymal stem cells (BMSCs) could restore the structure and function of injured tissues, but the homing and restorative effects of BMSCs on chemotherapy injured ovaries are still not clear. In this study, we found that granulosa cell (GC) apoptosis induced by cisplatin was reduced when BMSCs were migrated to granulosa cells (GCs) in vitro. Chemotherapy-induced POF was induced by intraperitoneal injection of cisplatin in rats. BMSCs labeled with enhanced green fluorescent protein (EGFP) were injected into the rats via the tail vein to investigate the homing and distribution of BMSCs in vivo. The number of BMSCs in the ovarian hilum and medulla was greater than in the cortex, but no BMSCs were found in the follicles and corpus lutea. In addition, the BMSCs treatment group’s antral follicle count and estradiol levels increased after 30 days, compared with the POF group. Hence, our study demonstrates that intravenously delivered BMSCs can home to the ovaries, and restore its structure and function in POF model rats.     

Effects of a gonadotropin-releasing hormone agonist on rat ovarian follicle apoptosis: regulation by epidermal growth factor and the expression of Bcl-2-related genes

The purpose of the present study was to evaluate the in vivo effect of the GnRH analogue leuprolide acetate (LA) on follicular development and apoptosis-related mechanisms in preovulatory ovarian follicles (POF) obtained from prepubertal eCG-treated rats. Serum progesterone and estradiol levels were measured, and a significant decrease in circulating estradiol levels was observed in the LA group, whereas serum progesterone levels remained unchanged. Ovarian histology revealed an inhibitory effect of LA treatment on the follicular development induced by eCG. After 48 h of LA treatment, the numbers of atretic and preantral follicles were increased as compared with controls, whereas the number of antral follicles had decreased. Cells undergoing DNA fragmentation were quantified by performing in situ 3' end labeling of DNA with digoxygenin-dUTP on ovarian sections. LA treatment caused an increase in the percentage of apoptotic cells in preantral and antral follicles. DNA isolated from these POF incubated 24 h in serum-free medium exhibited the typical apoptotic DNA degradation pattern. Treatment of follicles with epidermal growth factor (EGF) suppressed the spontaneous onset of DNA fragmentation, and a similar effect was observed in LA follicles. POF obtained from LA-treated rats showed no changes in Bcl-2 or Bax protein levels. However, a reduction in the Bcl-xL:Bcl-xS ratio was observed, with a greater decrease in Bcl-xL compared with Bcl-xS during the incubation, suggesting a lower stability of the Bcl-xL isoform in the LA group. These results indicate that in vivo GnRH agonist treatment produces an increase in the apoptosis process in POF from eCG-treated rats, and this effect is reversed in vitro by EGF. This GnRH analogue also reduced the stability of the Bcl-xL protein, thus interfering with follicular development by an as yet unknown mechanism.     

New strategy for in vitro activation of primordial follicles with mTOR and PI3K stimulators

It had been known for decades that primordial follicles in mammalian ovaries are assembled with definite numbers and represent the ovarian reserve throughout the reproductive life. Intra-oocyte PI3K/mTOR pathways have been indicated to play a central role on the activation of primordial follicles. Genetic modified mouse models with chronic activation of PI3K/mTOR signals in primordial oocytes showed premature activation of all primordial follicles and eventually their exhaustion. On the other hand, this may suggest that, unlike chronic activation of PI3K/mTOR, its acute activation in infertility would activate primordial follicles, permitting fertility during the treatment. Previously, PI3K stimulators were reported as a temporary measure to accelerate primordial follicle activation and follicular development in both mouse and human, and were applied in the treatment of infertility in premature ovarian failure (POF) patients. To address whether mTOR stimulators could play similar role in the process, we transiently treated neonatal and aged mouse ovaries with mTOR stimulators-phosphatidic acid (PA) and propranolol. Our results demonstrated the stimulators increased activation of primordial follicles and the production of progeny. Human ovarian cortex cubes were also treated with mTOR or/and PI3K stimulators in vitro. When they were used separately, both of them showed similar promotive effects on primordial follicles. Surprisingly, after joint-treatment with the 2 kinds of stimulators together, synergistic effects on follicular development were observed. Based on increased efficiency of follicular activation in humans, here we propose in vitro transient treatment with mTOR and PI3K stimulators as an optimized protocol for the application in different clinical conditions with limited follicle reserve.     

Human placenta‐derived mesenchymal stem cells inhibit apoptosis of granulosa cells induced by IRE1α pathway in autoimmune POF mice

Previous studies have shown that the ovarian failure in autoimmune‐induced premature ovarian failure (POF) mice could be improved by the transplantation of human placenta‐derived mesenchymal stem cells (hPMSCs); however, the protective mechanism of hPMSCs transplantation on ovarian dysfunction remains unclear. Ovarian dysfunction is closely related to the apoptosis of granulosa cells (GCs). To determine the effects of hPMSCs transplantation on GCs apoptosis, an autoimmune POF mice model was established with zona pellucida glycoprotein 3 (ZP3) peptide. It is reported that the inositol‐requiring enzyme 1α (IRE1α) and its downstream molecules play a central role in the endoplasmic reticulum (ER) stress‐induced apoptosis pathway. So the aim of this study is to investigate whether hPMSCs transplantation attenuated GCs apoptosis via inhibiting ER stress IRE1α signaling pathway. The ovarian dysfunction, follicular dysplasia, and GCs apoptosis were observed in the POF mice. And the IRE1α pathway was activated in ovaries of POF mice, as demonstrated by, increased X‐box binding protein 1 (XBP1), up‐regulated 78 kDa glucose‐regulated protein (GRP78) and caspase‐12. Following transplantation of hPMSCs, the ovarian structure and function were significantly improved in POF mice. In addition, the GCs apoptosis was obviously attenuated and IRE1α pathway was significantly inhibited. Transplantation of hPMSCs suppressed GCs apoptosis‐induced by ER stress IRE1α signaling pathway in POF mice, which might contribute to the hPMSCs transplantation‐mediating ovarian function recovery.     

Autoimmune targeted disruption of the pituitary-ovarian axis causes premature ovarian failure

Premature ovarian failure (POF) is characterized by amenorrhea and high serum levels of follicle-stimulating hormone (FSH). POF causes female infertility and represents a substantial women's health risk affecting 1% of women by age 40. Although ovarian autoimmunity has been associated with POF, the identity of ovarian Ags recognized is unknown. In this study, we show that autoimmune-targeted disruption of the pituitary-ovarian axis leads to POF. Immunization of SWXJ female mice with the p215-234 peptide derived from mouse inhibin-alpha activates CD4(+) T cells and induces experimental autoimmune oophoritis with a unique biphasic phenotype characterized by an early stage of enhanced fertility followed by a delayed stage of POF. Affected mice show high serum levels of inhibin-alpha-neutralizing Abs that prevent inhibin-mediated down-regulation of activin-induced pituitary FSH release. The loss of activin/FSH down-regulation leads to prolonged metestrus-diestrus, superovulation, increased numbers of mature follicles, increased offspring, accelerated depletion of primordial follicles, and ultimately premature infertility. Thus, inhibin-alpha-targeted experimental autoimmune oophoritis is initiated by CD4(+) Th1 T cells that stimulate B cells to produce inhibin-alpha-neutralizing Abs directly capable of mediating POF and transferring disease into naive recipients. Our inhibin-alpha autoimmune model of POF shows how premature infertility may develop in the context of elevated FSH levels thereby closely mimicking the hallmark features of human POF.     

Rictor/mTORC2 Pathway in Oocytes Regulates Folliculogenesis,and Its Inactivation Causes Premature Ovarian Failure

Molecular basis of ovarian folliculogenesis and etiopathogenesis of premature ovarian failure (POF), a common cause of infertility in women, are not fully understood. Mechanistic target of rapamycin complex 2 (mTORC2) is emerging as a central regulator of cell metabolism, proliferation, and survival. However, its role in folliculogenesis and POF has not been reported. Here, we showed that the signaling activity of mTORC2 is inhibited in a 4-vinylcyclohexene diepoxide (VCD)-induced POF mouse model. Notably, mice with oocyte-specific ablation of Rictor, a key component of mTORC2, demonstrated POF phenotypes, including massive follicular death, excessive loss of functional ovarian follicles, abnormal gonadal hormone secretion, and consequently, secondary subfertility in conditional knock-out (cKO) mice. Furthermore, reduced levels of Ser-473-phosphorylated Akt and Ser-253-phosphorylated Foxo3a and elevated pro-apoptotic proteins, Bad, Bax, and cleaved poly ADP-ribose polymerase (PARP), were observed in cKO mice, replicating the signaling alterations in 4-VCD-treated ovaries. These results indicate a critical role of the Rictor/mTORC2/Akt/Foxo3a pro-survival signaling axis in folliculogenesis. Interestingly, loss of maternal Rictor did not cause obvious developmental defects in embryos or placentas from cKO mice, suggesting that maternal Rictor is dispensable for preimplantation embryonic development. Our results collectively indicate key roles of Rictor/mTORC2 in folliculogenesis, follicle survival, and female fertility and support the utility of oocyte-specific Rictor knock-out mice as a novel model for POF.     

Optimization of the reconstruction of dermal papilla like tissues employing umbilical cord mesenchymal stem cells

Alopecia is not life threatening, but patients who undergo alopecia often experience severe mental stress. In addition, the number of individuals afflicted by alopecia has been increasing steadily. The most effective treatment of alopecia developed to date is auto hair transplantation. To overcome the limitations associated with current therapies for the treatment of alopecia, many researchers have attempted to revive hair follicles by in vitro culture of hair follicle cells and subsequent implantation in the treatment area. Previously, we demonstrated that umbilical cord-derived mesenchymal stem cells (UC-MSCs) could be isolated and expanded successfully from the Wharton’s Jelly. Cultureexpanded UC-MSCs formed aggregates similar to native dermal papilla (DP) in special media (DPFM) and reconstructed dermal papilla like tissues (DPLTs) could induce new hair follicles. The purpose of the present study was to optimize the reconstruction of DPLTs. As in the case of MSCs, when compared to differentiated cells, DPLTs require an additional step to induce differentiation into dermal papilla cells. However, it is necessary to use hepatocyte growth factor (HGF) in the differentiation step, which is relatively expensive. To reduce the expenses associated with cell therapy using MSCs, it is necessary to optimize this differentiation step. To accomplish this, we evaluated the effects of cell inoculation density and growth factors during differentiation.     

当归多糖通过AKT/FOXO3通路调节免疫性卵巢早衰小鼠内分泌功能

为探讨当归多糖(angelica polysaccharide,AP)对免疫性卵巢早衰小鼠内分泌功能的影响及分子机制,本研究将60只雌性BALB/c小鼠分为正常组、模型组、AP低剂量(100 mg/kg)、中剂量(200 mg/kg)、高剂量(400 mg/kg)组和补佳乐阳性组,以小鼠透明带多肽为抗原,皮下多点注射建立免疫性卵巢早衰(premature ovarian failure,POF)小鼠模型,灌胃相应药物,连续治疗4周后检测各组小鼠卵巢指数,检测各组小鼠血液SOD和MDA水平;通过ELISA检测各组小鼠卵巢组织IL-1β和IL-6水平,Western blotting检测各组小鼠p-AKT和FOXO3蛋白表达水平。与空白组相比,模型组小鼠卵巢指数显著降低,与模型组比较,AP低、中、高剂量组和阳性组小鼠卵巢指数显著升高,差异具有统计学意义(p<0.01)。与空白组比较,模型组小鼠血液SOD活性显著降低,MDA含量显著升高,与模型组比较,AP低、中、高剂量组和阳性组小鼠血液SOD活性显著升高,MDA含量显著降低。ELISA结果显示,与空白组比较,模型组小鼠卵巢组织IL-1β和IL-6含量显著升高,与模型组比较,AP低、中、高剂量组小鼠卵巢组织IL-1β和IL-6含量显著降低,差异具有统计学意义(p<0.01)。Western blotting结果显示,与空白组比较,模型组小鼠卵巢p-AKT和FOXO3蛋白表达显著降低,与模型组比较,AP低、中、高剂量组和阳性组小鼠卵巢p-AKT和FOXO3蛋白表达显著升高,差异具有统计学意义(p<0.01)。本研究结果说明,当归多糖AP激活AKT/FOXO3通路,抑制小鼠氧化应激,改善免疫性卵巢早衰小鼠内分泌功能,可在一定程度上抑制免疫卵巢早衰。     

Characterization of goose SPMS: Molecular characterization and expression profiling of SPMS in the goose ovary

Spermine synthase (SPMS), which converts spermidine into spermine, is essential for normal cell growth and development processes in humans and other mammals, but the molecular characterization and expression profiling of the SPMS gene remain undetermined in goose tissues and ovarian follicles. In this study, the SPMS cDNA sequence of the Sichuan white goose was cloned and analysed, and SPMS mRNA expression was profiled in various tissues and ovarian follicles. The results showed that the open reading frame of the SPMS cDNA sequence was 1092?bp in length, encoding 363 amino acids with a molecular weight of 41?kDa. Among all the examined tissues, SPMS expression was highest in the spleen and cerebrum and lowest in the breast and thigh muscles. SPMS expression in the F1 follicle was significantly higher than that in the POF (except for POF2) (P?<?0.05). Our results indicate that SPMS might play an important role in follicular development and ovulation.     

Histological evaluation of the effect of VEGF on auto-transplanted mouse ovaries

One of the most important factors affecting survival rate of ovarian follicles during transplantation period is proper vascular development. The objective of the study was to evaluate the effect of vascular endothelial growth factor (VEGF) on auto-transplanted ovarian tissue. Twenty-one-day-old female mice (n?=?30) transplanted as control group and 21-day-old female mice (n?=?40) were divided into 4 groups that were treated with 0.5, 1, 2 and 4?µg/mL of VEGF directly injected to auto-transplanted ovarian tissue. Twenty-one days after transplantation, mice were treated with 7.5 IU pregnant mare serum gonadotropin and human chorionic gonadotropin. Transplanted ovaries were removed and sections were prepared from transplanted tissues for staining. The most effective dosage of VEGF on transplanted tissue was determined over H&;E (hematoxylin and eosin) staining results. Slides were compared using TUNEL staining and CD31 assay for the most effective dosage. The percentages of preantral and antral follicles were not significantly different between transplanted group with 4?µg/mL VEGF and non-transplanted group. Lower apoptotic areas and higher CD31 expression were observed in transplanted ovaries treated with 4?µg/mL VEGF when compared to transplanted ovaries without VEGF treatment. VEGF positively affects the quality of ovarian tissue during transplantation. Survival rate of follicles and follicular development has improved with the effect of VEGF.     

Amniotic Fluid Stem Cells Prevent Follicle Atresia and Rescue Fertility of Mice with Premature Ovarian Failure Induced by Chemotherapy

Chemotherapy used to treat cancer may cause irreversible premature ovarian failure (POF). Of late, amniotic fluid stem cells (AFSCs) provide a novel source for regenerative medicine because of their primitive stage, low immunogenicity, and easy accessibility. In this study, we isolated AFSCs from transgenic mice that ubiquitously express enhanced green fluorescence protein (EGFP). These AFSCs exhibited morphologies, immunophenotypes, and mesoderm trilineage differentiation potentials similar to mesenchymal stem cells (MSCs). Further, AFSCs proliferated faster than MSCs and expressed OCT4, a marker for pluripotency. To investigate their potential in recovering fertility in POF model, AFSCs were transplanted into the ovaries of mice with POF six weeks post induction using chemotherapeutic drugs, busulfan and cyclophosphamide. AFSCs could rescue the reproductive ability of mice with POF by preventing follicle atresia and sustaining the healthy follicles. Notably, the transplanted AFSCs did not differentiate into granulosa and germline cells in vivo. After one month, the decreased numbers of transplanted AFSCs accompanied with the reduced beneficial effects indicated that the therapeutic efficacy were directly from AFSCs. These findings demonstrated the therapeutic effects of AFSCs and suggested the promise of AFSCs for treating infertility and POF caused by chemotherapy.     

Clinical analysis of human umbilical cord mesenchymal stem cell allotransplantation in patients with premature ovarian insufficiency

ObjectivePremature ovarian insufficiency (POI) is a refractory disease that seriously affects female fertility. Growing body of evidence has indicated mesenchymal stem cells (MSCs) as promising resources in regenerative medicine. In this study, we treated POI patients with umbilical cord‐derived MSCs (UCMSCs) and then investigated the restoration of ovarian function and clinical outcomes through follow‐ups.Materials and methodsSixty‐one patients diagnosed with POI participated in this study. UCMSCs were isolated and cultured according to GMP standards, and then transplanted to the patients’ ovary by orthotopic injection under the guidance of vaginal ultrasound. We monitored side effects, vital signs and changes in clinical and collected haematological and imaging parameters during the follow‐ups.ResultsAll patients showed normal clinical behaviour without serious side effects or complications relevant to the treatment. Transplantation of UCMSCs rescued the ovarian function of POI patients, as indicated by increased follicular development and improved egg collection. POI patients who experienced shorter amenorrhoea durations (<1 year) seemed to obtain mature follicles more easily after stem cell therapy, and patients with better ovarian conditions (pre‐operative antral follicles) were more likely to derive the better outcomes by UCMSC injection. Four successful clinical deliveries were obtained from POI patients after UCMSC transplantation, and all of these babies are developed normally.ConclusionsThe clinical trial result sugggests a possible therapy for POI by UCMSC transplantation.     

Development of antral follicles in cryopreserved cat ovarian tissue transplanted to immunodeficient mice

Ovarian cortex cryopreservation and xenotransplantation into immunodeficient mice represents a potential means for female germplasm conservation and an immediate model for investigation of folliculogenesis. The objectives of this study were to: (1) assess follicle survival after cryopreservation and transplantation of cat ovarian tissue into non-obese diabetic severely combined immunodeficient (NOD SCID) mice; and (2) evaluate the effects of gonadotropin treatments on follicular development in the transplanted tissue. Slices from the cat ovarian cortex were frozen and after thawing, transplanted under each kidney capsule of castrated male NOD SCID mice (eight xenografts in four mice). Sixty-two days after surgery, mice were randomly assigned (two per group) to gonadotropin-treated (eCG and hCG 88 h later) or control (saline-treated) groups. Twenty-four hours after the last injection, ovarian tissue was recovered and processed for histology. Fresh ovarian tissue from the same original source was similarly processed. Follicles were counted, measured, and classified as primordial, primary, secondary, or antral. Immunoreactive proliferating cell nuclear antigen (PCNA) stain was used to assess follicle viability. Microscopic examination revealed no evidence of necrosis or fibrosis. The grafts were well-vascularized, with follicles at all stages of development. Numbers of follicles in the transplanted tissue were markedly reduced compared to fresh tissue, with approximately 10% of follicles surviving freezing and transplantation procedures. Growing follicles positive for PCNA were found in all xenografts. Gonadotropin treatment did not alter the proportion of resting to growing follicles or mean follicle diameter by comparison with controls from untreated mice. By contrast, luteinization, but not ovulation, of antral follicles was observed only in grafts from treated mice. In summary, frozen-thawed cat ovarian cortex tissue not only survived xenotransplantation, it also contained follicles able to grow to antral stages. Exogenous gonadotropin treatment in this model resulted in luteinization of antral follicles but enhancement of follicular growth and ovulation did not occur.     

Recent achievements in in vitro culture and preservation of ovarian follicles in mammals

The mammalian ovary contains a large number of follicles that are in various developmental stages. The largest portion of them are primordial follicles. However, throughout the female reproductive lifespan only a small proportion of these follicles will produce oocytes competent to undergo successful maturation and ovulation. The rest of the ovarian oocytes (>99.9%) undergo atresia. It would be of great practical benefit to rescue some of these follicles by growing them in culture in order to provide an extra source of gametes. There is considerable interest in developing technologies that aim to produce fully-grown, developmentally competent oocytes from a pool of early developmental stages of follicles. Two methods have been used: 1/ long-term in vitro culture of either follicles or oocytes, and 2/ transplantation of ovarian tissue grafts. The development of efficient technologies may provide an additional source of oocytes for livestock production and reproduction in humans and rare or endangered species. The aim of this paper is to present a comprehensive review of recent achievements in the utilization of small ovarian follicles (primordial, preantral and early antral) by long-term in vitro culture and/or transplantation of ovarian tissue grafts (fresh and cryopreserved) in mammals including humans.     

The application of umbilical cord-derived MSCs in cardiovascular diseases

Transplantation of stem cells is a promising, emerging treatment for cardiovascular diseases in the modern era. Mesenchymal stem cells (MSCs) derived from the umbilical cord are one of the most promising cell sources because of their capacity for differentiation into cardiomyocytes, endothelial cells and vascular smooth muscle cells in vitro/in vivo. In addition, umbilical cord-derived MSCs (UC-MSCs) secrete many effective molecules regulating apoptosis, fibrosis and neovascularization. Another important and specific characteristic of UC-MSCs is their low immunogenicity and immunomodulatory properties. However, the application of UC-MSCs still faces some challenges, such as low survivability and tissue retention in a harmful disease environment. Gene engineering and pharmacological studies have been implemented to overcome these difficulties. In this review, we summarize the differentiation ability, secretion function, immunoregulatory properties and preclinical/clinical studies of UC-MSCs, highlighting the advantages of UC-MSCs for the treatment of cardiovascular diseases.     

Distribution of follicles in canine ovary – A simple and rapid method for counting follicles-

The distribution of follicles within canine ovarian cortex was evaluated to estimate follicular homogeneity. The analysis of follicular homogeneity prior to ovarian tissue transplantation limits the impact if follicular heterogeneity on experimental results. In this report, ovarian fragments from 14 immature bitches were embedded in OCT compound. Sections (5-μm-thick) were cut on a cryostat and stained with methylene blue. The mean number follicles ranged from 3.7 to 15.6/mm² in the 14 ovaries examined. The variance and distortion ranged from 2.05 to 144.30 and −2.09 and 2.01, respectively. The distribution of follicles was considered even, when the variance value was lower than 10 or between 10 and 16; and absolute value of distortion was inferior to 1. The distribution of follicles within ovarian cortex in 9 of 14 bitches was judged uneven. These results indicated that follicles were not homogeneously distributed within the ovarian cortex of the majority of bitches.