Cryopreservation of Human Ovarian Tissue

New and often aggressive treatment schemes allow the successful healing of many young patients with cancer, but the price the young women have to pay is high: many of them lose ovarian function and fertility. Due to the improved long-term survival of adolescents and young women with malignancies undergoing gonadotoxic chemotherapy, preservation of future fertility has been the focus of recent ubiquitarian interest. A feasible solution is the cryopreservation of ovarian tissue. Ovarian tissue, after thawing, can be used in three different ways: 1. grafted into its normal site (orthotopic); 2. grafted into a site other than its normal position (heterotopic), necessitating recourse to in vitro fertilization (IVF); 3. grown and in vitro matured in order to obtain metaphase II oocytes for an IVF program. It is believed that protein supplementation, in cryopreservation solution, is essential for improving ovarian tissue cryopreservation. The aim of this study was to evaluate the ultrastructural appearance of human ovarian tissue cryopreserved in 1.5 M 1,2 propanediol (PROH), 0.2 M sucrose using different protein sources: fetal calf serum (FCS), plasmanate or syntetic serum substitute (SSS). Fresh and frozen/thawed ovarian tissues were compared by transmission electron microscope (TEM), to evaluate the appearance of stromal and follicle cells as affected by different protein sources. Our data indicate that FCS is a better protein support for ovarian tissue cryopreservation when compared to SSS or Plasmanate. In addition the follicles are more resistant to the cryopreservation with respect to stroma.     

Risk and mechanism of glucose metabolism disorder in the offspring conceived by female fertility maintenance technology

Although female fertility maintenance technology (FFMT) provides an effective option for preserving fertility in patients with cancer suffering from fertility loss due to cancer treatment, previous studies have shown that the technique has certain potential risks and requires an assessment of the health status of the offspring since FFMT may lead to glucose metabolism disorder in offspring mice. The present animal study examined the glucose metabolism of adult mice offspring born from ovarian tissue cryopreservation and orthotopic allotransplantation. The mice were divided into three groups: normal, fresh ovary transplantation, and cryopreserved ovary transplantation. We recorded fasting blood glucose, glucose tolerance, and fasting serum insulin level for six months. Liver DNA, RNA, and proteins were extracted to detect the interaction between DNA methylation and Grb10 expression and insulin signaling pathway factors such as P-IGF1R, P-IRS2, P-AKT, and Grb10. Female recipient mice that received FFMT could successfully give birth after mating. The average litter size and total litter size of the cryopreserved and fresh groups showed marked differences compared with the normal group. Compared with the normal group, the fasting blood glucose and fasting serum insulin levels were higher in the cryopreserved and fresh groups. The mRNA and protein expressions of Grb10 were higher in the fresh and cryopreserved groups. Compared with the normal group, the DNA methylation status of four of the 11 sites of the Grb10 promoter was lower in the cryopreserved group. Grb10 overexpression inhibited the downstream phosphorylation protein factor expression (p-IGF-1R, p-IRS2, and p-Akt) of the IGF-1R signaling pathway. Female fertility maintenance technology (FFMT), including ovarian tissue cryopreservation (OTC), and orthotopic allotransplantation techniques might lead to glucose metabolism disorders in offspring mice.     

Evaluation of the hormonal function and histological features of heterotopic isogenic ovarian transplantation in rats

The purpose of the study was to determine the feasibility of preserving ovarian function after heterotopic transplantation by means of microvascular anastomosis of the transplanted vascular pedicles to a set of preselected vessels. Six groups of 10 Sprague-Dawley inbred rats were used in this study. Group I underwent bilateral ovariectomy operation and served as the ovariectomy control. Group II underwent bilateral ovariectomy followed by heterotopic isogenic ovarian implantation. Group III underwent bilateral ovariectomy and isogenic heterotopic ovarian transplantation by means of microvascular anastomosis. Group IV served as the laparotomy sham-operated control. Group V served as the ovarian donor for group II. Group VI served as the donor of the ovarian-kidney vascular pedicle complex for group III. Postoperative ovarian estradiol levels were measured, and histological characteristics were elucidated in groups I, II, III, and IV. The results demonstrated that the estradiol level of the transplantation group was comparable to that of the sham operation group and was significantly higher than that of the implantation group. Histologically normal ovarian architecture was observed in the sham group (IV) and also in the transplantation group (III). Altered architecture was observed in the implantation group (II). These findings indicate that extraabdominal heterotopic ovarian transplantation with microvascular anastomosis led to normal ovarian hormonal function and was effective in preserving oocyte production capacity.     

Human umbilical cord mesenchymal stem cell transplantation restores damaged ovaries

Ovarian injury because of chemotherapy can decrease the levels of sexual hormones and potentia generandi of patients, thereby greatly reducing quality of life. The goal of this study was to investigate which transplantation method for human umbilical cord mesenchymal stem cells (HUMSCs) can recover ovarian function that has been damaged by chemotherapy. A rat model of ovarian injury was established using an intraperitoneal injection of cyclophosphamide. Membrane‐labelled HUMSCs were subsequently injected directly into ovary tissue or tail vein. The distribution of fluorescently labelled HUMSCs, estrous cycle, sexual hormone levels, and potentia generandi of treated and control rats were then examined. HUMSCs injected into the ovary only distributed to the ovary and uterus, while HUMSCs injected via tail vein were detected in the ovary, uterus, kidney, liver and lung. The estrous cycle, levels of sex hormones and potentia generandi of the treated rats were also recovered to a certain degree. Moreover, in some transplanted rats, fertility was restored and their offspring developed normally. While ovary injection could recover ovarian function faster, both methods produced similar results in the later stages of observation. Therefore, our results suggest that transplantation of HUMSCs by tail vein injection represents a minimally invasive and effective treatment method for ovarian injury.     

The effect of increased dietary intake on superovulatory response to FSH in heifers

We have previously shown that the number of ovarian follicles <4 mm in diameter can be increased by enhanced dietary intake in heifers. This study investigated the effect of the same dietary treatment on superovulatory response. The estrous cycles of 24 mature Hereford x Friesian heifers were synchronized by a standard progesterone plus prostaglandin protocol. The animals were fed with either 100% (group M, n = 12) or 200% (group 2M, n = 12) maintenance requirements for a 3-week period. Starting from day 4 of the synchronized estrous cycle, all the animals were superovulated using a standard 4-day FSH regime followed by an injection of GnRH analogue (GnRHa) to induce ovulation. Rectal ultrasound scanning was carried out to assess ovarian follicular populations at the start of FSH treatment and on the day of GnRHa injection, and to determine the number of corpora lutea 5 days after GnRHa injection. The body weight (BW) and body condition score (BCS) were recorded weekly and plasma samples were collected throughout the experimental period. There were no differences in either BW or BCS between two groups at the start of the experiment. The BW and BCS were maintained during the experiment in the group M, whilst animals in the group 2M showed a non-significant (P > 0.05) increase in BW and BCS. Circulating concentrations of insulin were significantly (P < 0.01) higher in heifers from the group 2M throughout the controlled feeding period. The group 2M had significantly (P < 0.05) more follicles 2-4 mm in diameter at the start of FSH treatment and more (P < 0.01) follicles >9 mm in diameter on the day of GnRHa injection, when compared with the group M. Similarly, 5 days after GnRHa injection there were significantly (P < 0.01) more corpora lutea in the group 2M (18.1+/-2.2) than in the group M (10.6+/-3.0). In addition, plasma progesterone concentrations following GnRHa injection were significantly (P < 0.01) higher in heifers from the group 2M. In conclusion, these results confirm that increased dietary intake can enhance the recruitment of ovarian follicles in heifers. This treatment may provide a valuable approach to improving superovulatory response in cattle.     

Folliculogenesis Is Not Fully Inhibited during GnRH Analogues Treatment in Mice Challenging Their Efficiency to Preserve the Ovarian Reserve during Chemotherapy in This Model

As many chemotherapy regimens induce follicular depletion, fertility preservation became a major concern in young cancer patients. By maintaining follicles at the resting stage, gonadotropin-releasing hormone analogues (GnRHa) were proposed as an ovarian-protective option during chemotherapy. However, their efficacy and mechanisms of action remain to be elucidated. Mice were dosed with cyclophosphamide (Cy, 100–500mg/kg i.p) to quantify follicular depletion and evaluate apoptosis at different times. We observed a dose-dependent depletion of the follicular reserve within 24 hours after Cy injection with a mean follicular loss of 45% at the dose of 200mg/kg. Apoptosis occurs in the granulosa cells of growing follicles within 12 hours after Cy treatment, while no apoptosis was detected in resting follicles suggesting that chemotherapy acutely affects both resting and growing follicles through different mechanisms. We further tested the ability of both GnRH agonist and antagonist to inhibit oestrus cycles, follicular growth and FSH secretion in mice and to protect ovarian reserve against chemotherapy. Although GnRHa were efficient to disrupt oestrus cycles, they failed to inhibit follicular development, irrespective of the doses and injection sites (sc or im). Around 20% of healthy growing follicles were still observed during GnRHa treatment and serum FSH levels were not reduced either by antagonist or agonist. GnRHa had no effect on Cy-induced follicular damages. Thus, we showed that GnRHa were not as efficient at inhibiting the pituitary-gonadal axis in mice as in human. Furthermore, the acute depletion of primordial follicles observed after chemotherapy does not support the hypothesis that the ovary may be protected by gonadotropin suppression.     

Preservation of fertility in females treated for cancer

Advancements of diagnosis and treatment have substantially improved cancer survival rates in the last few decades. The increasing number of survivors focuses attention on long-term effects caused by cancer treatment and its impact on quality of life. Ovarian failure is one of the major sequelae of cytotoxic chemotherapy and/or radiotherapy in female children and reproductive-age women. Oncologists should address the patients about fertility preservation options before therapy. Embryo cryopreservation is the only well-established method for females in preserving fertility; however other strategies including ovarian suppression, ovarian transposition and cryopreservation of oocytes and ovarian tissue are still experimental. Patients need advice and to know which are the most practical options for them. This article reviews the available fertility preservation methods in women, and the related issues including normal physiology of the ovary, effect of anticancer therapy on fertility, role of the oncologist and ethics. We performed a MEDLINE search from 1971 to 2011 in a similar way as Jensen et al. 2011, using the following MeSH terms: antineoplastic agents; ovarian failure; premature; infertility, female; fertility preservation; child and cancer; reproductive technologies, assisted.     

Immature cryopreserved ovary restores puberty and fertility in mice without alteration of epigenetic marks

Background

Progress in oncology could improve survival rate in children, but would probably lead to impaired fertility and puberty. In pre-pubertal girls, the only therapeutic option is the cryopreservation of one ovary. Three births have been reported after reimplantation of cryopreserved mature ovary. Conversely, reimplantation of ovary preserved before puberty (defined as immature ovary) has never been performed in humans.

Methodology/Principal Findings

In order to analyze ovarian function, we performed transplantation using fresh or cryopreserved immature grafts in pre-pubertal or adult mice. Puberty as well as cyclic hormonal activity was restored. All follicle populations were present although a significant reduction in follicle density was observed with or without cryopreservation. Although fertility was restored, the graft is of limited life span. Because ex vivo ovary manipulation and cryopreservation procedure, the status of genomic imprinting was investigated. Methylation status of the H19 and Lit1 Imprinting Control Regions in kidney, muscle and tongue of offsprings from grafted mice does not show significant alteration when compared to those of unoperated mice.

Conclusions/Significance

These results demonstrate that immature ovarian grafting can restore spontaneous puberty and fertility. However, these data suggest that follicle depletion leads to premature ovarian failure. This study addresses the very important epigenetics issue, and provides valuable information to the study of ovarian transplantation suggesting that these procedures do not perturb normal epigenetics marks. These results are highly relevant to the reimplantation question of immature cortex in women.     

环磷酰胺辅助化疗对不同年龄乳腺癌患者卵巢功能的损伤情况及机制研究

目的:以月经变化情况及血清激素水平为观察指标,探讨环磷酰胺辅助化疗方案对绝经前不同年龄段乳腺癌患者卵巢功能的损伤情况及可能的机制研究.方法:收集2017年1月至2018年12月期间在我院接受环磷酰胺辅助化疗的绝经前乳腺癌患者77例.患者年龄范围为26-48岁,并按年龄分为两组:≤ 35岁组为16例;＞35岁组为61例....     

Intraovarian Transplantation of Female Germline Stem Cells Rescue Ovarian Function in Chemotherapy-Injured Ovaries

Early menopause and infertility often occur in female cancer patients after chemotherapy (CTx). For these patients, oocyte/embryo cryopreservation or ovarian tissue cryopreservation is the current modality for fertility preservation. However, the above methods are limited in the long-term protection of ovarian function, especially for fertility preservation (very few females with cancer have achieved pregnancy with cryopreserved ovarian tissue or eggs until now). In addition, the above methods are subject to their scope (females with no husband or prepubertal females with no mature oocytes). Thus, many females who suffer from cancers would not adopt the above methods pre- and post-CTx due to their uncertainty, safety and cost-effectiveness. Therefore, millions of women have achieved long-term survival after thorough CTx treatment and have desired to rescue their ovarian function and fertility with economic, durable and reliable methods. Recently, some studies showed that mice with infertility caused by CTx can produce normal offspring through intraovarian injection of exogenous female germline stem cells (FGSCs). Though exogenous FGSC can be derived from mice without immune rejection in the same strain, it is difficult to obtain human female germline stem cells (hFGSCs), and immune rejection could occur between different individuals. In this study, infertility in mice was caused by CTx, and the ability of FGSCs to restore ovarian function or even produce offspring was assessed. We had successfully isolated and purified the FGSCs from adult female mice two weeks after CTx. After infection with GFP-carrying virus, the FGSCs were transplanted into ovaries of mice with infertility caused by CTx. Finally, ovarian function was restored and the recipients produced offspring long-term. These findings showed that mice with CTx possessed FGSCs, restoring ovarian function and avoiding immune rejection from exogenous germline stem cells.     

Gonadotropin-Releasing Hormone for Preservation of Ovarian Function during Chemotherapy in Lymphoma Patients of Reproductive Age: A Summary Based on 434 Patients

Background

Gonadotropin-releasing hormone agonists (GnRHa) might play a role in preserving ovarian function in lymphoma patients by inhibiting chemotherapy-induced ovarian follicular damage. However, studies of its clinical efficacy have reported conflicting results.

Method

We conducted a meta-analysis to determine the effect of the preservation of ovarian function by administering GnRHa in young patients with lymphoma undergoing chemotherapy. Seven studies were identified that met inclusion criteria and comprised 434 patients assigned to GnRHa combined chemotherapy or chemotherapy alone.

Results

The incidence of women with premature ovarian failure (POF) demonstrated a statistically significant difference in favor of the use of GnRHa (OR=0.32, 95% CI 0.13-0.77). In addition, the final level of FSH in the GnRH group was significantly lower than control group. (MD= -11.73, 95% CI,-22.25- -1.20), and the final level of AMH in the GnRH group was significantly higher than control group (MD=0.80; 95% CI, 0.61–0.98). However, there was no statistically significant difference between treatment and the control groups in the incidence of a spontaneous pregnancy (OR=1.11; 95% CI, 0.55–2.26).

Conclusion

This meta-analysis suggests that GnRHa may be effective in protecting ovarian function during chemotherapy in lymphoma patients. More well-designed prospective studies are needed to carry out for further understanding of this topic.     

Cryogenic effect of antifreeze protein on transgenic mouse ovaries and the production of live offspring by orthotopic transplantation of cryopreserved mouse ovaries

The purpose of the present study was to evaluate the cryogenic effect of antifreeze protein (AFP) on transgenic mouse ovaries which is expressed AFP type III from Ocean pout and the production of live offspring by orthotopic transplantation of cryopreserved mouse ovaries. In this study, whole transgenic and nontransgenic mouse ovaries were vitrified with 20% DMSO and 20% EG in M2 medium supplemented with 0.5 M sucrose. All vitrified and toxicity control and fresh ovaries were transplanted orthotopically into ovariectomized recipients bilaterally. For fresh ovaries transplantation, 5 mice delivered litters of 18 and 19 live pups in first and second matings, respectively. For toxicity control of chemicals, 6 mice delivered litters of 22 and 23 live pups. For nontransgenic mouse ovaries (vitrified) transplantation, 7 mice delivered litters of 22 and 23 live pups. For transgenic mouse ovaries (vitrified) transplantation, 10 mice delivered litters of 35 and 37 live pups. Litter sizes from pups of freshly transplanted ovaries were not significantly different from AFP-transplanted transgenic ovaries but those from nontransgenic-transplanted ovaries were significantly different from the AFP-transplanted transgenic ovaries group (P < 0.05). In this study, for the first time, it was shown that the ovarian tissue of AFP transgenic mice was protected from cryopreservation by vitrification. These results demonstrate that a normal reproductive lifespan can be restored by orthotopic transplantation of AFP transgenic-vitrified ovary.     

Restoration of fertility after treatment for cancer

The late effects of chemotherapy and radiation treatment on fertility are an important issue for long-term survivors of cancer who may not have started or completed a family at the time of diagnosis. Attempts at protecting reproductive function using hormonal manipulation have proved largely unsuccessful and other strategies have to be considered. For men, semen cryopreservation allows subsequent artificial insemination of a female partner or ivf but cryopreserved semen is a finite resource, does not allow natural conception and is not an option for prepubertal boys. In an effort to overcome this, research is in progress to investigate whether testicular cells harvested and cryopreserved before the start of chemotherapy can be reintroduced to the testis after treatment and resume normal spermatogenesis. This has been achieved in a mouse model and the results of experimental protocols in men are awaited with interest. For women, harvested mature oocytes are only poorly tolerant of the freezing process although immediate in vitro fertilization and cryopreservation of embryos can be successful. An experimental technique of great interest is the harvesting and cryopreservation of ovarian cortex before the start of sterilizing treatment. In ewes, the reimplantation of autologous ovarian cortical tissue into surgically castrated animals has resulted in resumption of oestrus, conceptions after normal matings and the birth of live offspring. Recently, ovarian function has been re-established using a similar technique in a patient following treatment for Hodgkin's lymphoma, but so far pregnancy has not been reported.     

Development of infantile rat ovaries autotransplanted after cryopreservation by vitrification

We cryopreserved infantile rat ovaries by vitrification and assessed their viability by autotransplantation. Hemilateral ovarian transplantation was performed on rats on postnatal Days 10 to 12. The left ovary of each rat was dissected out, cryopreserved by vitrification using a modified vitrification solution (VS1), and then autotransplanted under the capsule of the right kidney. The right ovary of each rat was removed. For the control, the left ovary was dissected out from each rat and was immediately transplanted by the same procedure, without cryopreservation. Rats were nursed until weaning, and then the day of vaginal opening, estrous cyclicity from the day of vaginal opening until postnatal Day 84, and histology of ovarian grafts at postnatal Day 84 were examined. The time course of development of endocrine function of cryopreserved grafts was similar to that of fresh grafts. In ovarian transplants recovered on postnatal Day 84, antral follicles and corpora lutea (CL) were observed in addition to small follicles, although the number of antral follicles in cryopreserved grafts was smaller than in the fresh grafts. These results indicate that cryopreservation of ovarian tissue by vitrification can be used for the preservation of fertility and endocrine function of ovaries.     

Conséquences de la chimiothérapie anticancéreuse sur la fertilité de la femme

Sterility is a potential toxic effect of chemotherapy. This risk is well established for alkylating agents, but is less clearly defined for anthracyclines, methotrexate and fluorouracil and poorly defined for alkaloids, platinum, etoposide and taxanes. The main predictive factors for ovarian toxicity are the additive effect of cytotoxic drugs, the cumulative dose of each drug and the patient’s age. This effect of chemotherapy is evaluated on menstrual cycles, hormonal assays and the number of pregnancies observed in patient cohorts. Chemotherapy induces destruction of oocytes and granulosa cells. In mice, it has been shown that adriamycin may induce oocyte apoptosis, which can be prevented by modulation of cycle cell signalling (dysregulation of Bax gene or, on the contrary, expression of its antagonist gene Bcl-2 or inhibition of apoptosis with sphingosine-1-phosphate or caspase inhibitors). Clinical data in the literature are usually based on retrospective studies and are somewhat confused: global fertility after MOPP chemotherapy for Hodgkin’s disease is about 20%, adjuvant chemotherapy with CMF, F(A)C or TAC for breast cancer induces amenorrhea in 50% to 70% of cases, PVB or BEP chemotherapy for ovarian germ cell tumors has little effect on fertility when the uterus and one ovary can be preserved, and the majority of women treated with methotrexate, actinomycin D or various combinations for persistent trophoblastic disease remain fertile. Preservation of fertility is a major goal for cancer patients receiving chemotherapy: in vitro fertilization could preserve the couple’s fertility, but is usually not feasible as it would delay initiation of chemotherapy until after stimulation of ovulation; oocyte or ovarian tissue cryopreservation is at the stage of research; oral contraceptives have not been demonstrated to be effective to preserve ovarian function; gonadotropin releasing hormone (GnRH) agonists prevent cyclophosphamide toxicity in rat and monkey ovaries, and a few pilot clinical studies suggest that chemotherapy-induced amenorrhea could be prevented by administration of GnRH analogues simultaneously to chemotherapy, but randomised studies are necessary.     

促性腺激素释放激素类似物缓解环磷酰胺导致卵巢损伤的机制研究

目的:通过动物实验和细胞实验初步探索促性腺激素释放激素类似物在环磷酰胺暴露下保护卵巢功能的机制。方法:构建乳腺癌荷瘤小鼠36只,随机分成Control组,CTX组和CG(CTX+GnRHa)组,药物干预后麻醉下剥离肿瘤和子宫卵巢并称重,计数卵巢原始卵泡和生长卵泡数目,提卵巢蛋白进行Western blot检测抗缪勒氏管激素(Anti-Müllerian hormone,AMH)蛋白表达量,提取卵巢RNA进行qRT-PCR实验检测mRNA水平,心尖取血检测血清AMH水平。卵巢颗粒细胞加药处理36小时后,收细胞蛋白进行Western blot检测AMH蛋白表达量,提细胞RNA进行qRT-PCR检测mRNA水平,ELISA法检测细胞培养基上清中AMH水平。结果:成瘤后第21天,CTX组和CG组小鼠肿瘤质量无统计学差异(P>0.05)且均小于Control组(P<0.05);CTX组子宫和卵巢的总重量均显著低于Control组和CG组(P<0.01),卵巢内原始卵泡数量均低于Control组和CG组(P<0.001),生长卵泡数量无统计学差异(P>0.05)。动物实验,Western blot结果提示,CTX组卵巢AMH蛋白含量显著高于Control组或CG组(P<0.05);由ELISA结果提示,CTX组血清AMH蛋白浓度显著低于CG组(P<0.01)。细胞实验,由Western blot可见,CTX组AMH蛋白含量显著高于Control组和CG组(P<0.05);由ELISA实验可见,CG 750组和CG1000组培养基上清的AMH蛋白浓度均高于对应CTX剂量组(P<0.01)。无论在动物实验还是细胞实验中,各组AMH的mRNA表达水平均无统计学差异(P>0.05)。结论:在CTX化疗的同时运用GnRHa,可以在不干扰化疗疗效的前提下,通过减少CTX所致细胞内AMH潴留程度,增加细胞外和血清中的AMH浓度从而发挥保护卵巢储备的功能。     

Effects of Three Different Types of Antifreeze Proteins on Mouse Ovarian Tissue Cryopreservation and Transplantation

Background

Ovarian tissue (OT) cryopreservation is effective in preserving fertility in cancer patients who have concerns about fertility loss due to cancer treatment. However, the damage incurred at different steps during the cryopreservation procedure may cause follicular depletion; hence, preventing chilling injury would help maintain ovarian function.

Objective

This study was designed to investigate the beneficial effects of different antifreeze proteins (AFPs) on mouse ovarian tissue cryopreservation and transplantation.

Methodology

Ovaries were obtained from 5-week-old B6D2F1 mice, and each ovary was cryopreserved using two-step vitrification and four-step warming procedures. In Experiment I, ovaries were randomly allocated into fresh, vitrification control, and nine experimental groups according to the AFP type (FfIBP, LeIBP, type III) and concentration (0.1, 1, 10 mg/mL) used. After vitrification and warming, 5,790 ovarian follicles were evaluated using histology and TUNEL assays, and immunofluorescence for τH2AX and Rad51 was used to detect DNA double-strand breaks (DSBs) and repair (DDR), respectively. In Experiment II, 20 mice were randomly divided into two groups: one where the vitrification and warming media were supplemented with 10 mg/mL LeIBP, and the other where media alone were used (control). Ovaries were then autotransplanted under both kidney capsules 7 days after vitrification together with the addition of 10 mg/mL LeIBP in the vitrification-warming media. After transplantation, the ovarian follicles, the percentage of apoptotic follicles, the extent of the CD31-positive area, and the serum FSH levels of the transplanted groups were compared.

Principal Findings

In Experiment I, the percentage of total grade 1 follicles was significantly higher in the 10 mg/mL LeIBP group than in the vitrification control, while all AFP-treated groups had significantly improved grade 1 primordial follicle numbers compared with those of the vitrification control. The number of apoptotic (TUNEL-positive) follicles was significantly decreased in the groups treated with 1 and 10 mg/mL LeIBP. The proportion of τH2AX-positive follicles was significantly reduced in all AFP-treated groups, while the proportion of Rad51-positive follicles was significantly decreased in only the FfIBP- and LeIBP-treated groups. In Experiment II, after autotransplantation of OT vitrified with 10 mg/mL of LeIBP, the percentage of total grade 1 and primordial grade 1 follicles, and the extent of the CD31-positive area, were increased significantly. Moreover, the levels of serum FSH and the percentage of TUNEL-positive follicles were significantly lower in the LeIBP-treated than in the control group.

Conclusion

A supplementation with high concentrations of AFPs had protective effects on follicle preservation during OT vitrification-warming procedures. The group treated with LeIBP was protected most effectively. The beneficial effects of LeIBP were also observed after autotransplantation of vitrified-warmed OT. Further studies are necessary to determine the exact mechanism of these protective effects.     

人参皂甙Rb1对环磷酰胺所致卵巢早衰大鼠的保护作用

目的探讨人参皂甙Rb1对化疗所致卵巢早衰(Premature ovarian failure,POF)大鼠的Bcl-2及Bax基因表达的影响。方法选用30只2-3月龄具有正常动情周期雌性Wistar大鼠随机分3组,分别为对照组,治疗组和模型组。采用免疫组化和免疫印迹方法分别观察和半定量检测POF大鼠卵巢凋亡调节基因Bcl-2和Bax的蛋白表达情况,同时对比观察人参皂甙Rb1对其表达的影响。结果模型组卵巢颗粒细胞Bax蛋白的平均光密度值较对照组明显增高(P0.05);模型组Bcl-2蛋白的表达量较对照组明显下调(P0.05)。Rb1治疗后Bcl-2蛋白表达明显升高,Bax蛋白表达明显下调(P0.05)。结论 Rb1可能是通过下调bax蛋白水平减少卵巢颗粒细胞的凋亡,对化疗所致卵巢早衰起到治疗的作用,进而延缓卵巢衰老。     

High dose cisplatin compared with high dose cyclophosphamide in the management of advanced epithelial ovarian cancer (FIGO stages III and IV): report from the North Thames Cooperative Group.

A randomised study comparing cisplatin 120 mg intravenously with cyclophosphamide 2 g intravenously, each drug being given every month for six months followed by a low dose regimen for a further six months in responding patients, was carried out in 86 patients with advanced epithelial ovarian carcinoma (FIGO stages III and IV). Patients given cisplatin were found to have a longer median survival time than those given cyclophosphamide (19 months compared with 12 months) and a longer median duration of complete clinical response (18 months compared with eight months). The difference in disease free survival was statistically significant even after factors such as age, stage of disease, and the completeness of initial surgery had been taken into account. This study suggests that cisplatin is a more effective chemotherapeutic agent than cyclophosphamide for advanced ovarian cancer and should be the agent of choice in future trials comparing combination chemotherapy with a single agent.