Prevention and treatment of iatrogenic premature ovarian insufficiency:r interpretation of the first Chinese guideline on ovarian tissue cryopreservation and transplantation

In recent years,with the rapid development of medical research,cancer diagnosis and treatment technology have significantly improved young cancer patient’s survival rate.Anticancer therapy such as chemotherapy,radiotherapy,or hematopoietic stem cell transplantation can lead to premature ovarian insufficiency.The endocrine and reproductive function of the ovary is critical to women’s physical and mental health.Ovarian tissue cryopreservation and transplantation can protect not only female fertility but also preserve ovarian endocrine function.This paper interprets the guidelines for ovarian tissue cryopreservation and transplantation issued by the Chinese Society of Gynecological Endocrinology affiliated to the International Society of Gynecological Endocrinology.The purpose of this guideline’s interpretation is to promote more medical workers to understand the technology of ovarian tissue cryopreservation and transplantation,which can provide patients with more choices of fertility protection methods and improve their quality of life.

Morphological and ultrastructural changes in rat ovarian tissues after cryopreservation

Objective: The aim of the study was to observe and compare the effects of cryopreservation and thawing meth- ods on rat ovarian tissues. Methods: Twenty 5-6 weeks old SPF-SD female rats were randomly divided into two groups, with ten rats in each group. Freshly isolated ovaries saved as a control (group 1: fresh ovaries) in formalin-fixed or vitrified immediately after dissection (group 2: vitrified ovaries). Ovaries in vitrified group were processed into thin slices then cryo- preserved, stored in liquid nitrogen for 21 days, rapidly thawed and grossly examined. All of the collected ovaries underwent hematoxylin and eosin-stained paraffin serial sections and observed the microscopic evaluation in vitrified ovaries. Results: Grossly the vitrified ovaries turned pale color and the size was same as before freeze. The vitrified ovarian tissue had normal anatomical structures of cortex and medulla under the microscope and had no difference with the fresh control ovarian tis- sue. The number and distribution of the follicles were similar with the fresh ovarian tissue, but had smaller size and the gap between oocyte and the surrounding granulosa cells was increased. Few ooctyes were in irregular appearance however the morphology of follicular cells did not give a different appearance as compared to the fresh control ovarian tissue. Conclusion: Cryopreservation of ovarian tissues by vitrification method has some detrimental effect on the morphology of follicles but does not induce negative impact on the number, density and survival of the primordial ovarian follicles. However the whole follicle anatomical structures also had no significant changes.

Study on the Estrous Cycle Regularity of Cryopreserved Rat Ovarian Tissues after Heterotopic Transplantation

Objective: To evaluate the re-initiation of ovarian function in cryopreserved ovarian grafts by means of vaginal smear of transplant rats. Methods: A total of 40 SPF-SD female rats (5 - 6 week-old) were randomly divided into three groups (blank control, castration control and transplant group). Ovaries were removed by surgical procedure then after cryopreservation and thawing procedures the ovarian tissues pushed inside the back muscles gap in transplant group. On the first PO day, vaginal smear collection was daily initiated. After 30 days, the PO day when the estrous cycle was re-initiated was considered for analysis as well as the estrous days and the number of estrous cycles. Results: Normal control group had a regular estrous cycle, while the transplant group had an estrous cycle disorder within first 2 weeks and later 2 weeks after the transplantation while the duration of diestrus cycle lasted longer. At the same time, the castration group had lost the normal estrous cycle, keeping continue in the stage of diestrus. Conclusion: In transplanted animals the re-initiated ovarian function can be predicted with alteration between estrous and diestrus phases with predominant estrous irregularity. Moreover, short autotransplanted graft duration needs time to perfuse by new blood vessels and hormone secretions, so could not directly affect its target organs to function properly.

Modified vitrification method for cryopreservation of human ovarian tissues

Background Vitrification is a prospective technology in ovarian tissue cryopreservation, but it is still in an initial stage. This study was conducted to investigate a modified vitrification protocol for human ovarian tissue, which can be used as an alternative to preserve fertility for young women with cancer who have to undergo cytotoxic therapy and sterilization. Methods Ovarian tissue samples were collected from 15 patients and randomly allocated to groups of fresh, vitrification, and conventional slow freezing. A modified carrierless vitrification method was applied. The proportion of morphologically intact follicles in fresh ovarian tissues was compared with that in warmed/thawed tissues. The initial growth of the follicles and the concentrations of estradiol and progesterone were detected to determine the viability and endocrine function of the cryopreserved tissues. Results The proportion of morphologically intact primordial follicles in the fresh group （97.6%） was significantly higher than that in the other two groups （vitrification group 80.3% and slow-freezing group 72.6%, P〈0.001）. In both the vitrification and slow-freezing groups, estradiol and progesterone were secreted continuously during 2-week culture in vitro, the proportion of primary follicles were both significantly increased compared to the fresh group. No statistically significant differences existed between the two groups after cryopreservation in the proportion of both primordial and primary follicles, and the concentrations of estradiol and progesterone （P〉0.05）.Conclusion The modified vitrification method for cryopreservation of human ovarian tissues is effective, simple, and inexpensive.

Elective oocyte freezing for the preservation of fertility

Oocyte cryopreservation has recently emerged as an option for women to preserve their fertility for medical (e.g. treatable malignancy) or elective indications (e.g. advancing age). This report describes an IRB-approved study of over 200 oocyte cryopreservation cycles at one center. Patients presenting for oocyte cryopreservation (January 2005 to 2010) were analyzed for day 3 follicle stimulating hormone (FSH), basal antral follicle count (BAFC), gonadotropin usage and the number of oocytes retrieved and cryopreserved. New patient consultations were performed on 516 women, of whom 175 (34%) proceeded to initiate a total of 233 cryopreservation cycles. Twenty-four cycles were cancelled (10%) after starting follicular stimulation due to poor ovarian response or self-withdrawal of the patients. Patients whose cycles were cancelled demonstrated a higher Day 3 FSH and a lower BAFC than patients who completed cycles (p < 0.01). In the 209 completed cycles, the most important predictors of a successful cycle included BAFC (r = 0.36), FSH (r = –0.25) and age (r = –0.18) with the mean number of oocytes cryopreserved at 13.6 ± 8.8. Information about long-term fertility preservation must reach both patients and health care providers so that more women can be educated about the benefits of proactive early physiological reproductive assessment and possible interventions available.

卵巢组织冻存与移植技术新进展

卵巢组织冻存与移植技术不仅保护女性生育力,还可保存卵巢内分泌功能,有效防治医源性早发性卵巢功能不全,尤其适用于青春期前女童与亟需原发病治疗的女性。卵巢组织冻存的有效性与安全性已得到了广泛验证,目前的临床金标准为慢速冻存,而玻璃化冻存仅适用于研究。尽管该技术近年来发展迅速,但仍面临超低温冻存损伤、冷冻保护剂毒性、卵泡储备量的评估、冻存与移植的安全性等问题。本文针对以上问题的研究进展进行综述。

移植卵巢组织卵泡损伤机制及保护策略

卵巢组织冷冻保存及移植是青春期前女性及不能延迟放疗或化疗的恶性肿瘤患者唯一的生育力保存手段。目前,卵巢组织冷冻保存及移植效率仍低,卵巢组织移植后面临缺血缺氧风险,始基卵泡的异常激活和血供恢复后的缺血-再灌注损伤也造成移植卵巢组织卵泡大量损失。大量研究尝试通过添加某些药物减少卵泡在冷冻及移植过程中所受的损伤,延长卵巢移植患者内分泌功能及生殖功能持续的时间:如添加褪黑素、N-乙酰半胱氨酸、促红细胞生成素或其他抗氧化剂以减轻氧化应激,添加不同来源的间充质干细胞、碱性成纤维细胞生长因子、血管内皮生长因子、血管生成素2、促性腺激素以促进血运重建,添加抗米勒管激素、雷帕霉素以减少始基卵泡异常激活。本文重点阐述了卵巢组织移植后卵泡损伤的主要机制及减少移植卵巢组织卵泡损伤的方法,以期为提高卵巢组织移植效率提供参考。

Offspring from oocytes grown in frozen-thawed ovarian tissues transplanted to male and female bodies

Objective: There are few reports of live births from heterotopic transplantation of frozen-thawed ovarian tissue. The purpose of this study is to assess the follicular development in the frozen-thawed ovarian tissues following heterotopic transplantation in both female and male bodies.Methods: Cluster of differentiation 1 (CD1) mice (6-8 weeks) were used in this study as ovarian tissue donors and foster mothers for embryo transfer. Sperm from CD1 male mice were used for insemination by intracytoplasmic sperm injection (ICSI). Nude severe combined immunodeficiency mice (8 weeks) were employed as recipients of ovarian tissue transplantation. The frozen-thawed ovarian tissues were transplanted to 4 sites on each mouse, female and male, subcutaneously. After 3 months, both female and male mice were injected with 5.0 IU gonadotropins intraperitoneally. Post 48 hours of injection, the mouse was killed for ovarian transplant collection. Only fully grown oocytes with contacted cumulus cells (cumulus-oocyte complexes) were then selected for maturationin vitro.In vitro matured oocytes were inseminated with fresh sperm by ICSI, and the developed blastocysts were frozen using the vitrification method and stored until embryo transfer. After thawing, the thawed blastocysts were incubated for at least 2 hours before the transfer. The foster mice mothers mated with vasectomised male 3 days previously. Live birth was monitored at 19 days after transfer, and the resulted offspring was raised for fertility test.Results: The relatively high recovery rates of the transplanted ovarian tissues were collected in both frozen-thawed and fresh ovarian tissue transplants from both female and male bodies. The fully grown immature oocytes became maturein vitro and the fertilized zygotes developed to blastocyst stage. There are no differences between frozen-thawed and fresh ovarian transplants in term of oocyte quality and embryo development to blastocyst rates. Nineteen-day post-transfer, 3 foster mothers from the frozen-thawed ovarian tissue transplant group delivered 13 pups and the 4 foster mothers of the fresh ovarian tissue transplant group delivered 12 live pups. The produced offspring were normal in appearance and grew healthy and fertile.Conclusions: Our results attest that the follicles can survive and develop in the frozen-thawed ovarian tissues following the subcutaneous transplant to adult male mouse’s body regardless of basal endocrinal environment. Those fully grown oocytes can produce healthy and fertile offspring which will provide the possibility for further mechanistic understanding of endocrinology of folliculogenesis.

干细胞在卵巢组织冷冻与移植中应用的研究进展

干细胞在血液系统疾病中的临床应用已经较为成熟,并进行了广泛推广,例如骨髓造血干细胞移植治疗白血病等,挽救了很多白血病患者的生命。然而,干细胞在生殖医学领域仍处于较慢的初期发展阶段。虽然相对而言,干细胞在卵巢功能障碍的研究已经很多,但在卵巢冷冻与移植上有很多尚未开展且值得研究的方向。本文将从干细胞在卵巢组织冷冻保存和移植方面的最新研究进展进行综述,重点介绍不同来源干细胞改善损伤性卵泡存活的进展及其机制,以及在不孕症治疗和生育能力保存尤其是在卵巢冷冻移植方面的潜在临床应用价值。

女性癌症患者生育力保存技术

随着近年癌症患者生存率的不断提高以及相关辅助生殖技术的发展,针对女性癌症患者的生育力保存也成为了现阶段的研究热点。本文对近10年的文献研究中报道的女性生育力保存技术进行综述。包括卵巢组织冷冻与移植、卵巢组织体外激活技术、卵巢抑制、卵巢移位术、人工卵巢、未成熟卵母细胞冷冻和胚胎冷冻技术等生育力保存技术的有效性和安全性,并讨论比较了每种方法的优、缺点。以期将来能为女性癌症患者选择合适的生育力保存技术提供支持。

医源性早发性卵巢功能不全性激素水平的变化及对糖代谢的影响

目的研究医源性早发性卵巢功能不全(premature ovarian insufficiency,POI)性激素水平的变化及对糖代谢的影响。方法募集2022年1月到2024年4月就诊于首都医科大学附属北京妇产医院内分泌科的20~40岁的60例因造血干细胞移植(hematopoietic stem cell transplantation,HSCT)导致的医源性POI患者和60例特发性POI患者,一般资料包括身高、体质量、年龄,并计算体质量指数(body mass index,BMI),实验检测指标包括卵泡刺激素(follicle stimulating hormone,FSH)、黄体生成素(luteinizing hormone,LH)、雌二醇(estradiol,E 2)、皮质醇(cortisol,F)、总睾酮(total testosterone,TT)、游离睾酮(free testosterone,FT)、空腹血糖(fasting plasma glucose,FPG)及空腹胰岛素(fasting insulin,FINS)。结果医源性POI患者体质量、BMI、E 2、TT、FT低于特发性POI患者,差异有统计学意义(P均<0.05)。医源性POI患者的FSH、LH、FINS、FPG高于特发性POI患者,差异有统计学意义(P均<0.05)。医源性POI患者与特发性POI患者的年龄、身高和皮质醇相比,差异无统计学意义(P均>0.05)。医源性POI患者高胰岛素血症的比例明显高于特发性POI患者(P=0.025)。结论医源性POI患者性激素水平的降低和糖代谢异常均比特发性POI患者更加严重,可能原因是疾病治疗对卵巢功能的损伤更大。但医源性POI是可以预防的。卵巢组织冻存(ovarian tissue cryopreservation,OTC)可以同时保护生育力和卵巢内分泌功能,使医源性POI的防治从不可能变为可能,因此在接受放射治疗和化学药物治疗之前进行OTC十分有必要。

特纳综合征患者生育及妊娠管理的研究进展

妊娠是特纳综合征(Turner syndrome,TS)患者的关键问题,尽管4%~7%的TS患者有自然妊娠可能,但大多数女性需要借助卵母细胞捐献和生育力保护。由于卵巢功能减退通常发生在TS患者的生命早期,且妊娠可加重TS孕产妇基础心血管疾病进展或恶化的风险,如主动脉夹层或破裂等致死性并发症。因此,TS女性应该在孕前、妊娠期间和产后接受全面的心血管评估和多学科团队管理,以确保TS女性良好的生育结果。卵巢组织冻存技术是一种具有特定益处、适应证的个性化保护策略,有可能成为TS患者保护生育能力的重要工具。本文主要综述TS患者生育及妊娠管理的研究进展。

卵巢组织冻存移植保存肿瘤患者生育力的研究进展

肿瘤的放化疗提高了患者的生存率,但是会导致卵巢损伤。肿瘤治疗后的生育力损伤会严重降低患者的生活质量。卵巢组织冻存移植是需要立即放化疗年轻女性保存生育力的唯一可靠选择。其中,卵巢组织冻存最常用的方法是玻璃化冷冻法,该法可有效保存卵巢组织;卵巢组织移植则是在患者的肿瘤治疗结束后将冻存组织复苏后进行自体原位或异位移植以恢复卵巢生理功能。近年来,卵巢组织冻存移植作为一种有效的保存肿瘤患者生育力的方法并被广泛应用,它可以有效且安全地保护肿瘤患者的生育力,未来拥有巨大的发展潜力。

An experimental study on autologous transplantation of fresh ovarian tissue in sheep

Objective:To investigate current autologous transplantation methods and sites of ovarian tissues in sheep.Methods:Sheep ovaries were resected.Ovarian cortices were sliced and transplanted orthotopically into the ovarian mesangial latum and heterotopically into the greater omentum and under groin skin.The grafts were removed two months after transplantation and examined to evaluate the survival of follicles(hematoxylin-eosin staining)and help determining feasible graft sites and transplantation methods.Results:Graft nodules were found in the transplanted sites.HE staining of the grafts showed that multiple primordial follicles were able to survive in the grafts on both sides of the ovarian mesangial latum,the right side of the greater omentum,and the left inguinal subcutaneous tissue.Secondary or cystic follicles were found in almost all of the grafts.Conclusion:The ovarian mesangial latum,the greater omentum and the inguinal subcutaneous tissue can be used as autologous transplantation sites,where sheep ovarian tissue can survive and the follicles grow and develop in good condition.

两种冷冻保护剂玻璃化冷冻小鼠卵巢的比较研究

目的:探讨两种玻璃化冷冻保护剂对小鼠卵巢组织学和功能的影响。方法:将23只4周龄ICR雌鼠随机分为新鲜卵巢移植组(6只)、去势组(5只)、EG40组(6只)和ED20组(6只)。EG40组和ED20组分别应用乙二醇(EG)和联合应用EG与二甲基亚砜(DMSO)玻璃化冷冻小鼠卵巢组织,一周后解冻,将一部分复苏卵巢组织自体移植入小鼠肾被膜下,另一部分组织行组织学观察。移植术后5d开始观察所有小鼠的动情周期,一个月后处死小鼠,对存活的卵巢组织行组织学观察。结果:EG40组、ED20组和新鲜卵巢移植组小鼠动情周期出现率均为100%,出现动情周期的天数分别为10.2±1.2d、8.0±0.9d和6.8±1.0d,EG40组动情周期出现天数明显多于新鲜卵巢移植组(P<0.01);ED20组与新鲜卵巢移植组差异无显著性(P>0.05)。移植存活的卵巢组织内可见不同发育阶段的卵泡,形态正常,但冻融卵巢组织内卵泡数量比新鲜组织少。结论:联合应用玻璃化冷冻保护剂EG和DMSO对小鼠卵巢组织学和功能的影响较小。

人类卵子体外成熟及其胚胎冷冻复苏研究

目的:探讨取卵周期准备、卵泡发育状态、培养液成分对未成熟卵的获取率、体外成熟率、受精率、胚胎质量、胚胎移植后成功率及其胚胎冷冻复苏移植的影响。方法:19例进行卵子体外成熟的不孕不育患者为研究对象。其中1例为自然周期,14例为促性腺激素(Gn)诱导排卵周期,4例常规控制性促排卵(COS)周期;分别以TCM 199或人输卵管液(HTF)为基础培养液,进行卵子体外成熟培养。结果:当取卵时卵泡大小不均一且最大卵泡直径≥12 mm时,获卵率、受精率和胚胎质量下降;TCM 199组优质胚胎的形成率显著高于HTF组(P<0.01)。IVM胚胎经冷冻复苏、胚胎移植能获得与常规IVF周期相似的妊娠成功,出生后代健康、无畸形。结论:在IVM周期取卵时卵泡大小不均一且最大卵泡直径≥12 mm时,将影响胚胎着床率和累积妊娠率。TCM 199优于HTF培养液,能提高未成熟卵体外成熟后受精卵所形成胚胎的发育能力。IVM胚胎经冷冻复苏-胚胎移植能获得与常规IVF周期相似的妊娠成功,出生后代健康、无畸形。

两种冷冻方法对人大块卵巢组织卵泡活性的影响

目的探讨两种冷冻方法冻融人类大块卵巢组织的冷冻效果及对卵泡活性的影响。方法收集15例人卵巢组织,每例修剪成3块卵巢组织(约15 mm×15 mm×2mm),分别随机分配到新鲜对照组(A组)、玻璃化冷冻组(B组)、两步法冷冻组(C组),组织学分析3组卵泡形态学变化,并进行卵巢组织体外培养后测定培养液上清中雌激素、孕激素浓度,免疫组织化学染色测定培养后的卵巢组织细胞核增殖相关抗原Ki67及B细胞淋巴瘤/白血病-2基因(Bcl-2)表达水平,评估卵泡增殖及凋亡活性。结果 A组正常形态卵泡比率(91.9%)显著高于B组(71.3%)和C组(82.9%),差异有统计学意义(P<0.05),C组高于B组,差异有统计学意义(P<0.05)。C组的始基卵泡和初级卵泡的正常形态率分别为86.8%、54.4%,均分别高于B组73.8%、44.4%,差异有统计学意义(P<0.05)。同一时期3组激素测定结果差异不显著(P>0.05)。C组Ki67的阳性率为73%,分别显著高于A组50%和B组55%(P<0.05),A组低于B组(P<0.05)。Bcl-2阳性率,A组为57%和C组61%相比无差异(P>0.05),但B组42%阳性率分别低于A组和C组(P<0.05)。结论人体大块卵巢组织冻融后仍具有活性,并且两步法冻存效果优于玻璃化冷冻法。

人卵巢组织两种超低温冻存方法的研究

【目的】探索两种人卵巢组织冷冻方案用于生殖细胞保存的效果。【方法】活检卵巢组织来自15名经知情同意的手术患者。组织被随机分配到新鲜组、玻璃化冷冻组和慢速冷冻组。观察和比较各组卵巢组织冻融后的原始卵泡形态完整率,并通过体外培养系统测定培养液中的雌二醇和孕酮水平,观察和比较冻融后卵巢组织内分泌功能。【结果】本研究中新鲜组、慢速冻融组和玻璃化冻融组的原始卵泡形态完整率分别是97.6%、72.6%和80.3%。两冻融组与新鲜组相比明显下降(P<0.001),而在两冻融组间差异并无统计学意义(P=0.237)。在体外组织培养期间,两种冻融卵巢组织都能持续分泌雌二醇和孕酮,两组间差异无统计学意义(P>0.05)。【结论】本研究中两种人类卵巢组织超低温冷存的方法是可行的,可适应不同需要。

人卵巢组织冻存与移植研究进展

随着癌症治疗学的发展,癌症病人的生存率有了很大的提高。但性腺毒性治疗对肿瘤病人的生育力造成了不可逆的影响。有效的保护肿瘤病人的生育力,提高她们的生殖健康水平,是医疗工作者面临的重要问题。卵母细胞冻存、胚胎冻存、卵巢组织冻存等技术的发展是为女性生育力保护提供了可能。本文将简述目前常用的生育力保护方法,重点介绍人卵巢组织冻存与移植技术在生育力保护方面的重要作用。

以细胞筛为载体的玻璃化冷冻和慢速程序化冷冻保存人卵巢组织的效果比较

目的比较以细胞筛为载体的玻璃化冷冻与慢速程序化冷冻用于人卵巢组织冷冻的效果。方法人卵巢组织取皮质切块后,随机分为3组:新鲜组织对照组(F组)、慢速程序化冷冻组(S组)及以细胞筛为载体玻璃化冷冻组(V组)。卵巢组织冷冻复苏后,固定切片后行苏木素-伊红染色,观察卵泡形态;使用TdT介导的dUTP缺口末端标记技术,观察卵泡凋亡情况;部分卵巢组织体外培养,隔日采集培养液后检测雌二醇(E2)浓度。比较三组卵泡正常形态率、卵泡凋亡率及E2浓度。结果与F组原始卵泡正常形态率(91.1%)相比,V组原始卵泡正常形态率(88.1%)无显著差异(P>0.05),而S组原始卵泡正常形态率(79.6%)显著下降(P<0.001);V组初级卵泡正常形态率(74.2%)与S组(73.6%)相似,但两组均低于F组(89.0%)(P<0.05);凋亡检测中,3组凋亡率无显著差异(P>0.05);体外培养2周,各组E2浓度持续上升,F组E2浓度显著高于S组、V组(P<0.001),而S组与V组E2浓度无显著差异(P>0.05)。结论以细胞筛为载体的玻璃化冷冻能较好地保存人卵巢组织,复苏后组织体外培养后,还可持续分泌E2。