程序化冷冻对人始基卵泡与初级卵泡保存效果的影响

目的:探讨程序化冷冻对人卵巢组织内的始基卵泡与初级卵泡形态与凋亡的影响。方法:采用慢速程序化冷冻保存人的卵巢皮质,采用组织形态学、电镜及原位凋亡检测观察冷冻前后的始基与初级卵泡的形态学变化及凋亡情况。结果:冷冻前后人正常形态的始基卵泡比例无显著变化,而冷冻后形态正常的初级卵泡的比例较新鲜组显著下降(P<0.05)。冷冻后的初级卵泡内线粒体肿胀,胞浆及线粒体出现空泡化,而始基卵泡的超微结构保持良好。冷冻前后2种卵泡的凋亡率比较无差异。结论:程序化冷冻对人初级卵泡的形态损伤严重,对始基卵泡保存较好。     

卵巢囊肿与多囊卵巢皮质中卵泡密度分析及其冷冻保存

目的:探索来自卵巢囊肿及多囊卵巢综合征(polycystic ovary syndrome,PCOS)卵巢皮质中卵泡的分布特征,并观察冷冻对卵巢组织形态学的影响。方法:收集23例卵巢囊肿(卵巢囊肿组)及8例PCOS(PCOS组)的卵巢皮质,分析卵巢皮质中的卵泡密度;同时冷冻部分组织,观察冷冻前后各级卵泡的分布及形态学改变。结果:PCOS组中的卵泡密度明显高于卵巢囊肿组,但各卵泡囊肿组间的卵泡密度差异无显著性。冷冻后各组中卵泡分布以及形态正常的始基与初级卵泡比例,与冷冻前比较差异无显著性;而冷冻前后PCOS组织中初级卵泡比例显著高于卵巢囊肿组,形态正常的始基卵泡比例明显低于卵巢囊肿组。结论:卵巢囊肿与PCOS患者的卵巢皮质可作为人卵巢组织冷冻保存的标本来源。     

4种冷冻-解冻方法对家兔卵巢组织形态学的影响

目的:探讨适宜的卵巢组织冻存方案。方法:采用PROH(A2组)及DMSO(B2组)慢速程序化冷冻和DMSO+PROH(C2组)及DMSO+EG(D2组)玻璃化冷冻方法,冻存家兔卵巢组织,解冻复苏后,以相应的4组新鲜组织为对照(A1-D1组),做HE染色,行组织形态学分析。结果:A1-D1组始基卵泡的形态正常率分别为90.1%、91.5%、91.8%、92.2%,其相对应的A2-D2组分别下降为67.6%、69.7%、70.5%、80.1%,差异均有统计学意义(P均<0.05)。冷冻组中A2组始基卵泡形态正常率最高,与B2、C2、D2组比,差异有显著性(P<0.05)。C2、D2组间比,差异无显著性(P>0.05)。各冷冻组合并后形态正常率始基卵泡为72.7%,初级卵泡为55.7%,两者比较有统计学差异(P<0.05)。4个冷冻组中均可见卵巢组织结构受损的表现。结论:4种冷冻解冻方法对卵巢皮质中各级卵泡及卵巢组织结构均造成一定程度的损害,使各级卵泡的形态正常率明显下降,卵巢间质细胞连接变得疏松;PROH慢速程序化冷冻法明显优于DMSO法及玻璃化法,较适合卵巢组织中始基卵泡的保存;冻存卵巢组织对初级卵泡的影响大于始基卵泡。     

荷人卵巢上皮癌裸鼠冻融卵巢组织移植的效果评价

目的:获取人卵巢上皮癌裸鼠原位移植瘤癌旁正常卵巢组织,经安全筛查并冻融后移植至去势裸鼠体内,探讨移植后效果,为临床应用提供依据。方法:将人卵巢上皮癌OVCAR3细胞种植于裸鼠皮下以获取瘤源,并进行卵巢原位移植,建立卵巢癌原位移植瘤模型,解剖裸鼠获取癌旁正常卵巢组织。癌旁组:取筛选后的癌旁卵巢组织进行玻璃化冷冻,复苏后分别进行皮下及原位移植,各20例;对照组:同龄正常裸鼠卵巢组织,皮下移植及原位移植各20例;去势裸鼠组:20只同龄去势裸鼠;正常卵巢组:20只同龄正常裸鼠。移植12周后,分析各组裸鼠卵巢组织内卵泡形态及激素分泌功能。结果:40只人卵巢上皮癌裸鼠原位移植瘤模型中共获取35份活检正常的癌旁卵巢组织,获取率87.5%(35/40)。玻璃化冷冻前后卵巢组织中卵泡形态及各级卵泡比例均无显著差异(P>0.05),癌旁冷冻组织和癌旁新鲜组织的异常卵泡比例差异无统计学意义(P>0.05),冷冻组织以初级卵泡及次级卵泡等窦前卵泡为主。癌旁组皮下移植组织存活率80%(16/20),对照组皮下移植组织存活率90%(18/20),癌旁组原位移植组织存活率90%(18/20),对照组原位移植组织存活率95%(19/20)。移植后组织内卵泡以次级卵泡及窦状卵泡为主,各级卵泡的形态及构成比和未移植的同龄正常裸鼠卵巢相似(P>0.05);癌旁组卵泡数明显低于对照组(P<0.05)及正常卵巢组(P<0.01);皮下移植和原位移植组织内的各级卵泡数差异无统计学意义(P>0.05)。癌旁组卵泡刺激素水平明显低于去势裸鼠组,而雌二醇水平明显高于去势裸鼠组(P<0.01);癌旁组卵泡刺激素水平明显高于对照组(P<0.05)及正常卵巢组(P<0.01),而雌二醇水平明显低于对照组(P<0.05)及正常卵巢组(P<0.01);皮下移植和原位移植的卵泡刺激素水平和雌二醇水平差异无统计学意义(P>0.05)。结论:癌旁卵巢组织冻融移植后有正常卵泡发育及激素分泌功能;皮下移植和原位移植均可取得较好的效果;癌旁卵巢组织冻融移植有望作为卵巢上皮癌患者治疗后恢复卵巢内分泌功能的有效手段。     

自制冷冻环人卵巢组织玻璃化冷冻的可行性研究

目的探讨应用自制冷冻环进行人卵巢组织玻璃化冷冻的效果。方法2008年5月至9月在华中科技大学同济医学院附属同济医院收集5例人卵巢组织标本,以乙二醇、二甲基亚砜、蔗糖作为冷冻保护剂,自制冷冻环为载体进行玻璃化冷冻。并观察卵巢新鲜组织和冻融后组织始基卵泡和初级卵泡的形态学变化、凋亡情况、超微结构变化及体外培养内分泌功能。结果新鲜及冻融后组织形态正常卵泡比值分别是89.46±4.94、84.47±4.66,无统计学意义(P>0.05)。凋亡面积分别是(0.07±0.02)%、(0.10±0.05)%,无明显增加(P>0.05)。超微结构无明显改变。新鲜组织及冻融后组织体外培养上清液雌二醇含量分别(2549.73±711.87)pmol/L、(2514.87±714.66)pmol/L,无统计学意义(P>0.05)。结论自制冷冻环可作为一种便宜、方便、可行的人卵巢组织低温保存的可替代方法。     

冷冻保存对人卵巢组织雌二醇分泌功能的影响

目的:探讨冷冻保存对人卵巢组织E2分泌功能的影响。方法:收集20例卵巢良性肿瘤手术患者的正常卵巢皮质,随机分为新鲜对照组和玻璃化冷冻组,冻融后行体外培养,比较冻融前、后人卵巢组织的内分泌功能。结果:①在体外培养期间,所有卵巢组织均能持续分泌E2。新鲜对照组E2分泌水平始终高于冷冻组,培养前10d,差异有统计学意义(P<0.05);培养12d之后,组间E2水平无统计学差异(P>0.05)。②体外培养初期E2分泌水平较平稳,随培养时间延长E2水平呈波动性变化。结论:人卵巢组织冷冻解冻后仍具有内分泌功能,随着培养时间的延长,新鲜组与冷冻组间E2分泌水平可能无差异。     

两种冷冻方法对人类卵巢组织卵泡形态和组织增殖活性的影响

目的:探讨玻璃化冷冻和慢速冷冻何者更适于冻存人卵巢组织。方法:将10例因卵巢良性囊肿剔除术获取的人卵巢皮质组织切成薄片后随机分配到新鲜卵巢组(A组)、玻璃化冷冻组(B组)和慢速冷冻组(C组),通过光学显微镜和透射电子显微镜观察比较卵泡形态变化,免疫组织化学检测组织细胞增殖细胞核抗原(PCNA)表达变化。结果:A、B、C组中形态正常的原始卵泡比例分别占71.4%、70.1%、52.3%;形态正常的初级卵泡比例分别占76.0%、43.5%、31.8%;C组中形态正常的原始卵泡比例和初级卵泡比例均明显低于A、B组(P<0.05);B组形态正常的原始卵泡比例和初级卵泡比例与A组相比无统计学差异(P>0.05)。A、B组中形态正常的原始卵泡超微结构无明显改变,但B组中初级卵泡和C组中原始卵泡和初级卵泡的超微结构存在一定程度的改变。PCNA阳性表达主要见于卵母细胞、颗粒细胞和卵巢组织间质细胞,3组中均有PCNA表达,且表达无统计学差异。结论:玻璃化冷冻较慢速冷冻对人卵巢组织影响小,是一种较适宜的人卵巢组织冷冻保存方法。     

兔卵巢组织玻璃化冷冻的实验研究

目的:探讨玻璃化冷冻法保存兔卵巢组织的效果。方法:随机将25只新西兰雌兔分为对照组(5只)、慢速冷冻组(10只)和玻璃化冷冻组(10只),比较各组冻融前后卵巢组织学、超微结构、卵泡凋亡(原位末端标记法,TUNEL)和子宫系膜内移植后卵巢功能的恢复情况。结果:新鲜组织、慢速冷冻复苏组织和玻璃化冷冻复苏组织中正常形态卵泡比例分别为87.36%、81.96%和82.72%,两冷冻组正常卵泡比例均低于对照组,差异有统计学意义?P(0.05),但玻璃化冷冻组与慢速冷冻组差异无统计学意义(P>0.05)。3组间卵泡凋亡比率分别为21.4%、13.5%和17.1%,差异无统计学意义(P>0.05);3组移植后兔动情周期出现率均为100%,动情周期出现天数差异无统计学意义?P>0.05);移植存活的卵巢组织内可见各级形态正常的卵泡发育。结论:玻璃化冷冻可有效保存卵巢组织的结构和功能,是一种简单、可行的兔卵巢组织冷冻保存法。     

抗冷冻蛋白Ⅲ减少家兔卵巢组织冷冻损伤的效果观察

目的:评价抗冷冻蛋白Ⅲ(AFPⅢ)对玻璃化冷冻家兔卵巢组织的影响。方法:收集家兔卵巢30只,随机分为新鲜卵巢组、添加AFPⅢ玻璃化冷冻组(AFPⅢ终浓度为500 ng/ml)和常规玻璃化冷冻组,各组10只,解冻后分析各组卵巢的组织学结构、卵泡形态正常率、卵巢组织超微结构、卵母细胞凋亡率及卵泡存活率。结果:新鲜卵巢组的卵泡形态正常率(91.6%)、卵泡存活率(81.75%)显著高于两冷冻组(P0.01),卵母细胞凋亡率(12.0%)显著低于两冷冻组(P0.01);添加AFPⅢ玻璃化冷冻组卵泡形态正常率(77.5%)、卵泡存活率(45.31%)显著高于常规玻璃化冷冻组(分别为62.1%、37.25%)(P0.01),卵母细胞凋亡率(25.8%)显著低于常规玻璃化冷冻组(41.2%)(P0.01)。结论:家兔冷冻卵巢组织的卵泡形态正常率、卵泡存活率显著低于新鲜组织,冷冻保护剂中加入AFPⅢ可减少家兔卵巢组织冷冻损伤。     

卵巢组织冷冻技术在女性生育力保存中的应用

卵巢是雌性动物的重要生殖器官,富含许多不同发育阶段的卵泡,是一种天然的卵泡池。原始卵泡对冷冻保护剂的耐受性要高于生长卵泡,而卵巢皮质是原始卵泡的聚集地,因此卵巢皮质组织冷冻保存是一种更安全、有效、极具开发潜能的女性生育力保存方式。卵巢组织冷冻可以一次性保存大量的配子。然而,目前卵巢组织冷冻技术还是一个试验性的技术,临床应用面临很多困难和阻碍。本文将从卵巢组织冷冻技术、移植、临床应用及发展方向作一综述。     

Ovarian tissue cryopreservation and transplantation: scientific implications

After fresh or frozen ovary transplantation, FSH levels return to normal, and menstrual cycles resume by 150 days, coincident with anti-Müllerian hormone rising to higher than normal levels. AMH then returns to well below normal levels by 240 days, remaining as such for many years with nonetheless normal ovulation and fertility. To date, 20 babies have been born in our program from 11 fresh and 13 cryopreserved ovary transplant recipients with a live baby rate of over 70 % (11 babies from fresh and 9 from frozen). Globally, over 70 live births have been reported for both fresh and frozen ovary transplants with an approximate 30 % live birth rate. Given the rapid rise of AMH after the fall of FSH, with a subsequent AMH decrease with retention of ovarian function, it is tempting to speculate the existence of a shared mechanism controlling primordial follicle recruitment, fetal oocyte meiotic arrest, and recruitment in the adult ovary. With the massive recruitment of primordial follicles observed after human ovarian cortical tissue transplantation, which subsides to an extremely low recruitment rate, we will discuss how this phenomenon suggests a unifying theory implicating ovarian cortical tissue rigidity in the regulation of both fetal oocyte arrest and recruitment of follicles in the adult ovary. As the paper by Winkler-Crepaz et al. in this issue demonstrates, our in vivo results are consistent with the in vitro demonstration that primordial follicles in the fetal cortex are “locked” in development, resulting in meiotic arrest, which spares the oocytes from being rapidly lost all at once (Winkler-Crepaz et al., J Assist Reprod Genet, 1). Winkler-Crepaz et al. demonstrate that follicle loss after ovarian cortex transplantation is unlikely due to ischemic apoptosis, but rather from a “burst” of primordial follicle recruitment. In vivo, primordial follicles are normally resistant to further development or activation to prevent oocyte depletion. The dense fibrous ovarian cortex, through as yet unresolved mechanisms, arrests the further continuation of meiosis and also prevents a sudden depletion of all resting follicles in the adult ovary. Intrinsic tissue pressure is released after cortical tissue transplantation, temporarily resulting in a rapid follicle depletion. These results are consistent with the observation that once the ovarian reserve is reduced in the graft, the rate of recruitment diminishes and the ovarian tissue exhibits a relatively long duration of function.     

Blastocyst Development After Cryopreservation and Subcutaneous Transplantation of Mouse Ovarian Tissue

Purpose To investigate follicle survival and developmental potential with IVF of cryopreserved, subcutaneously transplanted mouse ovarian tissue.Methods Fresh and frozen mouse ovarian tissue was autologously transplanted into subcutaneous tissue. Two weeks after the transplantation, the morphology and histology of the fresh and frozen grafts were compared. Superovulation and IVF was performed to evaluate the fertility potential of the frozen ovarian graft.Results Both fresh and frozen grafts of ovarian tissue survived in 14 of 16 mice (88%). Morphologically, both types of grafts resembled fresh ovarian tissue and contained follicles at all stages of folliculogenesis. A total of 73% of follicles in fresh grafts and 62% in frozen grafts survived after transplantation compared with fresh ovarian tissue. Sixteen ICR mice underwent superovulation. A total of 56 oocytes from antral follicles were recovered from the subcutaneously transplanted cryopreserved ovarian tissue. Fourteen (25%) oocytes were in metaphase II stage, 6 were fertilized by IVF, and 2 progressed to the blastocyst stage.Conclusions Cryopreservation and subcutaneous transplantation of ovarian tissue provides a possible means of fertility preservation. The main loss of follicles occurred during grafting rather than during freezing and thawing.     

Follicular growth in fresh and cryopreserved human ovarian cortical grafts transplanted to immunodeficient mice.

OBJECTIVE: To investigate follicle growth in fresh and cryopreserved human ovarian cortical grafts transplanted to immunodeficient mice. STUDY DESIGN: Fresh or frozen-thawed human ovarian cortex was grafted subcutaneously or under the kidney capsule of 43 mice (35 nude mice and eight SCID mice), 14 of which were non-stimulated controls, 21 injected intra-peritoneally with gonadotrophins during 2 weeks and eight injected during 3 months. Follicle count was compared by Chi-square. RESULTS: Proportions of primordial follicles were significantly lower in grafts than in the tissue before transplantation in gonadotrophin-stimulated mice (37% versus 79%), but not in non-stimulated mice (51% versus 74%). Proportions of primary and secondary follicles were increased after transplantation indicating early follicular growth. One antral follicle was observed in a graft in a mouse stimulated for 3 months. CONCLUSION: Primordial follicles in fresh or frozen-thawed human ovarian cortex transplanted under the kidney capsule or subcutaneously can grow and are responsive to hormonal stimulation. Condensation: Primordial follicles in fresh and cryopreserved human ovarian cortical grafts can initiate growth after transplantation to immunodeficient mice     

Expression of inhibitor proteins that control primordial follicle reserve decreases in cryopreserved ovaries after autotransplantation

Purpose

Even with 86 live births reported globally so far, the mechanism of primordial follicle loss following autotransplantation of the frozen-thawed ovarian tissue needs further evaluation. Pten, Tsc1, p27, and Amh are the inhibitor proteins that play crucial roles in suppressing the transition from the primordial follicle to primary state, maintaining the primordial follicle reserve. In this study, we aimed to evaluate whether the expression patterns of these proteins change and it may be related to the global primordial follicle loss after autotransplantation of the frozen-thawed ovarian tissue.

Methods

Four groups were established in rats: fresh-control, frozen/thawed, fresh-transplanted, and frozen/thawed and transplanted. After slow freezing and thawing process, two ovarian pieces were transplanted into the back muscle of the same rat. After 2 weeks, grafts were harvested, fixed, and embedded into the paraffin block. Normal and atretic primordial/growing follicle count was performed in all groups. Ovarian tissues were evaluated for the dynamic expressions of the Pten, Tsc1, p27, and Amh proteins using immunohistochemistry, and H-score analyses were done.

Results

Ovarian tissue cryopreservation does not change the expression patterns of inhibitory proteins that control ovarian reserve. Both in fresh and frozen/thawed autotransplanted groups, the expression of inhibitory proteins and Amh decreased significantly in primordial follicles and in growing follicles, respectively. In control group and in frozen/thawed group, primordial follicle counts were similar but decreased by almost half in both fresh-transplanted and frozen/thawed and transplanted groups.

Conclusions

One of the causes of primordial follicle loss after transplantation of ovarian graft may be decreased expression of the inhibitory proteins that guard the ovarian reserve and transplantation itself seems to be the major cause for disruption of inhibitory molecular signaling. Our findings highlight important molecular aspects for future clinical applications for fertility preservation in humans.

     

Cryopreservation of female germ cells and ovarian tissues for fertility preservation

To preserve the fertility of patients who undergo chemotherapy and/or radiotherapy, procedures for cryopreservation of female germ cells have been investigated. Cyropreservation methods differ according to follicle stage because the mammalian ovary contains a large number of oocytes at different growth stages. Follicles at very early stages, for example the primordial and primary stages, are usually cryopreserved within ovarian cortical tissue because they need surrounding somatic cells for subsequent development. In contrast, fully-grown oocytes in Graafian follicles are cryopreserved without any other cells at the metaphase II stage. Recently, ultra-rapid cooling was incorporated into cryopreservation procedures for human ovaries. In this review, we describe oocyte freezing, the development of ultra-rapid cooling systems for ovarian tissues, freezing of human ovaries, and ovarian transplantation.     

Expression of transforming growth factor-alpha, epidermal growth factor, and epidermal growth factor receptor in follicles of human ovarian tissue before and after cryopreservation

OBJECTIVE: To study the expression of transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), and EGF receptor in follicles of human ovarian tissue. DESIGN: A retrospective, controlled comparative study. SETTING: In vitro fertilization laboratory of a university hospital. PATIENT(S): Fifteen women with regular menstrual cycles who underwent laparoscopy and the biopsy of ovarian tissue. INTERVENTION(S): Paraffin sections were prepared from ovarian tissues, followed by immunohistochemical staining of TGF-alpha, EGF, and EGF receptor. MAIN OUTCOME MEASURE(S): Immunostaining for TGF-alpha, EGF, and EGF receptor in follicles of fresh and frozen ovarian tissues. RESULT(S): Immunoreactivities for TGF-alpha and EGF receptor were observed simultaneously in the oocytes of primordial, primary, preantral, and antral follicles. Strong staining for TGF-alpha and EGF receptor was present in thecal cells. The TGF-alpha and EGF receptor was also expressed in some granulosa cells of primary to antral follicles. The EGF only stained weakly in the oocytes of primordial and primary follicles and in thecal cells. There was no difference in staining patterns for TGF-alpha, EGF, and EGF receptor between fresh and frozen ovarian tissues. CONCLUSION(S): The TGF-alpha and EGF receptor was expressed in primordial to antral follicles, indicating a role of TGF-alpha in regulating follicular development through binding to the EGF receptor. Freeze-thawing did not substantially alter immunoreactivites for TGF-alpha, EGF, and EGF receptor in frozen ovarian tissue.     

Follicle development following cryopreservation of human ovarian tissue

Despite the recent increase in human ovarian tissue banking, there has been little progress in establishing whether follicles within this tissue are viable and capable of function following cryopreservation. Two methods to assess growth and developmental potential of cryopreserved tissue are evaluated; (1). isolated follicle culture and (2). xenografting of tissue into a host animal. Development of numerous antral follicles following xenografting of cryopreserved tissue indicates that the cryopreservation procedure can preserve the developmental competence of primordial follicles.