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。     

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。     

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。     

Vitrified human ovaries have fewer primordial follicles and produce less antimüllerian hormone than slow-frozen ovaries

Slow-freezing and vitrification methods of human ovarian tissue cryopreservation were compared in terms of primordial follicle count and in vitro antimüllerian hormone (AMH) and estradiol production. Compared with fresh and slow-frozen ovaries, vitrified ovaries contained statistically significantly fewer primordial follicles and produced statistically significantly less AMH in vitro. Estradiol production from slow-frozen and vitrified ovaries was similar but statistically significantly lower than from fresh cultured strips.     

Cryopreservation of human ovarian tissue: effect of spontaneous and initiated ice formation

This investigation compared conventional freezing of human ovarian tissue using either spontaneous or initiated ('seeded') ice formation. Biopsies of ovarian tissue were obtained from women with indications for chemotherapy or radiotherapy. Small pieces of experimental tissue were randomly distributed into three groups that were then subjected to different treatments prior to culture in vitro for 16 days: the control group, no treatment, cultured immediately after biopsy (group 1); cryopreservation/thawing with spontaneous ice formation (group 2); and cryopreservation/thawing with initiated ice formation (group 3). Follicle viability and hormonal activity were then evaluated. There was no significant difference between groups regarding the concentration of oestradiol 17-beta in the culture supernatant, whereas progesterone concentration was significantly (P < 0.05) higher in group 1 compared with group 2 or 3. There was a significant (P < 0.05) difference in primordial and primary follicle density between all of the groups (group 1 having the highest and group 2 having the lowest) and group 2 had significantly (P < 0.05) fewer normal grade follicles than the other two groups. For optimal cryopreservation of human ovarian tissue, the protocol of conventional freezing should therefore include a step of initiated ice formation.     

Efficacy of ovarian tissue cryopreservation in a major European center

Purpose

To evaluate the effect of cryopreservation and thawing of ovarian tissue from oncological patients opting for fertility preservation on ovarian tissue viability.

Methods

In this prospective cohort study, the ovarian tissue viability before and after cryopreservation and thawing was measured for 25 newly diagnosed oncological patients who had their ovarian tissue cryopreserved. Outcome measures were follicle integrity (histology), follicle viability (Calcein viability assay), steroid hormone production (estradiol and progesterone production in vitro) and overall tissue viability (glucose uptake in vitro). This study was conducted at a Cryobank for storage of ovarian tissue in a university hospital.

Results

Cryopreserved/thawed ovarian tissue showed a decreased glucose uptake when compared to tissue that had not been cryopreserved. In addition, a diminished E2 and P4 production was observed after cryopreservation and thawing, despite the fact that numbers of viable follicles as determined by the Calcein viability assay were comparable. Histological examination revealed a higher percentage of degenerated follicles after cryopreservation and thawing.

Conclusions

Ovarian tissue cryopreservation and thawing impairs the viability of ovarian tissue in oncological patients opting for fertility preservation.     

Apoptosis of human ovarian tissue is not increased by either vitrification or rapid cooling

This study evaluated the incidence of morphological changes, as assessed by light microscopy, and apoptosis in vitrified and rapidly cooled human ovarian tissue. Apoptosis was assessed 30 min and 24 h after warming using transmission electron microscopy, terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and DNA fragmentation, as determined by gel electrophoresis. The results showed no significant changes in morphology, chromatin condensation, DNA fragmentation or TUNEL-positive cells in follicles attributable to cryopreservation or exposure to the cryoprotectant solutions alone. In conclusion, the cryopreservation protocols did not affect the incidence of apoptosis and either protocol could be an alternative to slow cooling of ovarian tissue.This study evaluated the incidence of morphological changes, as assessed by light microscopy, and apoptosis in human ovarian tissue cryopreserved using two different methods, i.e. vitrification and rapid cooling. Apoptosis was assessed in tissue 30 min and 24 h after warming using transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and DNA fragmentation as determined by gel electrophoresis. The results showed no significant changes in morphology, chromatin condensation, DNA fragmentation or TUNEL-positive cells in follicles attributable to cryopreservation or exposure to the cryopreservation solutions alone. In conclusion, the cryopreservation protocols did not affect the incidence of apoptosis in human ovarian tissue and either protocol could be an alternative to slow cooling for the preservation of ovarian tissue.     

Impact of the cryopreservation technique and vascular bed on ovarian tissue transplantation in cynomolgus monkeys

Purpose

The aim of this study was to determine the best combination in terms of cryopreservation techniques and vascular bed preparation before grafting in order to obtain functional ovarian tissue after transplantation.

Methods

Five cynomolgus monkeys were used. Strips from 10 ovaries were cryopreserved, 5 by vitrification (V), and 5 by slow-freezing (SF). Pieces of fresh ovarian tissue were used for controls. After 1 month, the strips were autografted to two different vascular beds, healed (HB) or freshly decorticated (FDB), constituting four study groups: SF-HB, SF-FDB, V-HB, and V-FDB. These were compared to fresh tissue. After 6 months, the ovaries were removed and several parameters analyzed: follicle quality, stage, density, proliferation, apoptosis, functionality, vascularization, and fibrosis. Mixed effect linear regression models were built to assess the impact of cryopreservation and vascular bed preparation on ovarian tissue viability and functionality. p values were adjusted for multiple testing using the Benjamini-Hochberg method, and q values < 0.20 were considered significant in order to achieve a 20 % false discovery rate.

Results

Compared to fresh tissue, no difference was observed in the percentage of morphologically normal follicles, while a significant increase was noted in the follicle proliferation rate (41 %, q = 0.19), percentage of antral follicles (12 %, q = 0.14), and number of vessels per area (3.3 times, q = 0.07) in the V-FDB group.

Conclusions

Vitrification associated with FDB vascular bed preparation is the best combination to obtain functional autografted ovarian tissue. Further studies are nevertheless required, with confirmed pregnancies and live births before introducing the procedure into clinical practice.

Electronic supplementary material

The online version of this article (doi:10.1007/s10815-015-0542-y) contains supplementary material, which is available to authorized users.     

Follicular morphology after ovarian cortex cryopreservation in the rabbit doe(Oryctolagus cuniculus)

OBJECTIVE: To compare the effects of two cryoprotective agents (DMSO and 1,2-PROH) used at two concentrations (1,5 and 2 M) on the morphology of small ovarian cortex follicles in doe. MATERIALS AND METHODS: Ovarian cortexes (n=40) were frozen in TCM199+10% FCS medium added to 1.5 or 2 M of DMSO or 1,2-PROH. Two controls were realized (fresh and frozen without cryoprotectant). The equilibration in cryoprotective solutions before freezing, and the elimination of the cryoprotective agents after thawing, was performed step by step. The effects induced by cryopreservation were evaluated by histological examination. RESULTS: Fresh ovarian tissue showed 68.6% of intact follicles. After freezing, only 1.5 M of 1,2-PROH preserved 48.0% of normal follicles, with no significant difference compared to the fresh control. The proportion of follicles without morphological defect observed after cryopreservation with DMSO was significantly reduced (respectively 28.8 and 34.8% for 1,5 and 2 M of DMSO). DISCUSSION AND CONCLUSIONS: Our results suggest that 1,2-PROH is a more effective cryoprotectant than DMSO, for the cryopreservation of doe ovarian cortex. These results differ from those that were obtained for other species, credibly because of a higher fragility of the ovarian tissue of the doe. Nevertheless, this species is an interesting animal model which allows rapid results after cryopreserved ovarian tissue graft.     

Effects of supra-zero storage on human ovarian cortex prior to vitrification–warming

This study investigated the effects of supra-zero storage on ovarian cortex of 12 premenopausal patients before cryopreservation. Fresh ovarian tissue (control) was either vitrified immediately or stored at 4°C for 24 h or 48 h before vitrification. This study assessed malondialdehyde release during storage and the capacity to synthesize oestradiol in culture, follicle morphology and the proportion of apoptotic follicles subsequent to vitrification–warming. The malondialdehyde concentration in the storage medium increased significantly in a time-dependent manner (P = 0.029). Oestradiol release during tissue culture decreased statistically significantly in both storage groups compared with the immediate vitrification group (P = 0.043). The proportion of high-quality follicles was significantly different between the fresh group and the two storage groups (24 h, P < 0.01; 48 h, P < 0.001). The proportion of apoptotic follicles was significantly different between the fresh and immediate vitrification groups (P < 0.05). The 24-h and 48-h groups were not different compared with the other groups. This study provides evidence that storage at supra-zero conditions has detrimental effects on the human ovarian cortex. Storage duration should therefore be limited to a minimum to prevent potential damage.     

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。     

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。     

Attempts to improve human ovarian transplantation outcomes of needle-immersed vitrification and slow-freezing by host and graft treatments

Purpose

To investigate if needle-immersed vitrification or slow-freezing yields better implantation results for human ovarian tissue and which method benefits more when combined with the “improvement protocol” of host melatonin treatment and graft incubation with biological glue?+?vitamin E?+?vascular endothelial growth factor-A.

Methods

Human ovarian tissue was preserved by needle-immersed vitrification or slow-freezing and transplanted into immunodeficient mice, either untreated (groups A and C, respectively) or treated with the improvement protocol (groups B and D, respectively). Grafted and ungrafted slices were evaluated by follicle counts, apoptosis assay and immunohistochemistry for Ki67 and platelet endothelial cell adhesion molecule (PECAM).

Results

Follicle number in the recovered grafts was limited. The number of atretic follicles was significantly higher after vitrification with/without the improvement protocol and slow-freezing than that after slow-freezing?+?the improvement protocol. Stroma cell apoptosis was the lowest in the group D. PECAM staining showed a peripheral and diffuse pattern in the group D (mostly normal follicular morphology) and a diffuse pattern in all other groups (few follicles, mostly atretic), with significantly higher diffuse levels in the vitrification groups. Ki67 staining was identified in all normal follicles. Follicles did not survive transplantation in the vitrification groups.

Conclusions

Ovarian sample preparation with slow-freezing?+?the improvement protocol appears to yield better implantation outcomes than needle-immersed vitrification with/without the improvement protocol. The real quality of frozen tissue can be assessed only after grafting and not after thawing/warming.

     

Mouse Ovarian Tissue Cryopreservation Has Only a Minor Effect on In Vitro Follicular Maturation and Gene Expression

Purpose : To establish a protocol for ovarian tissue cryopreservation which can retain fertility potential after thawing and to evaluate the impact of cryopreservation on development and gene expression during folliculogenesis. Methods : A controlled randomized study in a clinical and academic research setting in a university medical center was conducted to study cryopreservation and in vitro maturation (IVM) of mouse ovarian follicles. Preantral follicles isolated from either fresh (Group A) or cryopreserved (Group B) murine ovarian tissues were used to test their fertility potential by in vitro culture–in vitro maturation (IVC-IVM). Expression of Graafian follicles derived from both groups were detected by DNA microarray techniques for comparison. Results : Although there were no significant differences in IVM outcomes and follicular gene expression between the two experimental groups, cryopreservation appears to induce the expression of heat shock proteins, DNA-damage-inducible protein 45 and death-related apoptosis genes (i.e., Fas and Fas-ligand). Conclusion : Cryopreservation may trigger biological events not amenable to normal cell function and follicular development. However, neither follicular development nor gene expression was dramatically changed after cryopreservation. These data suggest that although our current cryopreservation techniques yield competent follicles and mature oocytes, subtle changes observed in gene expression imply that the present cryopreservation techniques need to be further refined.     

Clinically applied procedures for human ovarian tissue cryopreservation result in different levels of efficacy and efficiency

Purpose

Different protocols are being used worldwide for the cryopreservation of human ovarian tissue for fertility preservation purposes. The efficiency and efficacy of the majority of these protocols has not been extensively evaluated, possibly resulting in sub-optimally cryopreserved ovarian tissue. To address the impact of this issue, we assessed the effects of two clinically successful human ovarian tissue slow-freezing cryopreservation procedures on the quality of the cryopreserved tissue.

Methods

To differentiate between cryopreservation (_C) versus thawing (_T) related effects, four combinations of these two (A and B) very different cryopreservation/thawing protocols (A_CA_T, A_CB_T, B_CA_T, B_CB_T) were studied. Before and after cryopreservation and thawing, the percentage of living and morphologically normal follicles, as well as the overall tissue viability, was assessed.

Results

Our experiments revealed that the choice of the cryopreservation protocol noticeably affected the overall tissue viability and percentage of living follicles, with a higher viability after protocol B_C when compared to A_C. No statistically significant differences in tissue viability were observed between the two thawing protocols, but thawing protocol B_T required considerably more human effort and materials than thawing protocol A_T. Tissue morphology was best retained using the B_CA_T combination.

Conclusion

Our results indicate that extensive and systematical evaluation of clinically used protocols is warranted.

     

Cryopreservation of human ovarian tissue using the silver closed vitrification system

Purpose

The aim of this study is to evaluate the feasibility of using a hand-made silver container for the cryopreservation of human ovarian cortex.

Methods

Human ovarian cortex tissues were vitrified using an open vitrification system (OVS) of needle immersed vitrification (NIV) and two closed vitrification systems (CVS) of a plastic vial (plastic CVS) and a silver container (silver CVS). Outcomes of vitrification were evaluated morphologically and histologically by in vitro culture and xenotransplantation. The apoptosis of primordial follicles was assessed by TUNEL staining. The production of E₂ and P₄ was examined by a chemiluminescent immunoassay. Blood vessels were visualized with CD31 staining.

Results

Compared with the fresh ovarian cortex tissue, ovarian cortex tissues that were vitrified using the three different carriers and then warmed showed significantly reduced percentages of normal primordial follicles, viability of primordial follicles, E₂ and P₄ levels during in vitro culture and decreased amounts of blood vessels. However, much better outcomes were obtained with NIV and silver CVS than with plastic CVS, based on the better morphology and viability of primordial follicles, higher E₂ and P₄ production during an in vitro culture, and greater numbers of blood vessels after xenografting. Importantly, the outcomes of ovarian cortex cryopreservation with silver CVS were similar and comparable to those with NIV.

Conclusions

The hand-made silver container as a CVS is a promising carrier for the cryopreservation of the human ovarian cortex.

     

The hormonal micromilieu and oocyte status in the stimulated in vitro fertilization cycle

FSH, LH, PRL, estradiol-17 beta and progesterone were determined in 651 follicular fluids of 173 patients treated by ovarian stimulation for IVF. The stimulation was performed according to 5 different schemes: clomiphene/HCG, HMG (Pergonal)/HCG, HPG (Anthrogon)/HCG, clomiphene/HPG/HCG, Folistiman (heterologeous pituitary gonadotropin)/HCG. The mean levels of hormones of all follicles of each stimulation scheme were determined and differences between the groups were estimated by Student's t-test and the x-square-test. Additionally, in a hierarchy of follicles made depending on the follicular fluid volume the hormonal levels were compared between different rank numbers of one stimulation group and between different groups of stimulation. The maturation of oocytes judged by a maturation index and their ability for cleavage in culture after insemination was investigated in relation to the hormonal content of the follicular fluid. Stimulation by gonadotropins (Pergonal, Anthrogon, Folistiman) led to an decreased mean level of follicular steroids. This was related to an increased part of follicles poor in steroids after stimulation by gonadotropins. Within the follicular population follicles stimulated by clomiphene/HCG had a reduction of the levels of estradiol in higher rank numbers, but there were no clear evidences for such a reduction in the other stimulated groups. In all stimulation groups a significant reduction of progesterone levels was observed in higher rank numbers of follicles. Oocytes with a high maturation index mainly derived from follicles rich in progesterone. After insemination, development of oocytes in culture was compatible even with very high or low levels of hormones. There was no relation of the levels in FSH and LH to cumulus expansion and levels of estradiol of the follicular fluid. There was also no clear correlation between the levels of prolactin in follicular fluid and the cleavage rate.     

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.     

Comparison of two freezing protocols in an open freezing system for cryopreservation of rat ovarian tissue

AIM: To compare two freezing protocols in an automatic open-vessel freezing system for cryopreservation of rat ovarian tissue. METHODS: Ovarian tissue was transplanted heterotopically into the neck muscle, either without cryopreservation (group 1, n = 6) or with cryopreservation after equilibration with 1.5 mol/L dimethyl sulfoxide and propanediol (protocol A, group 2, n = 6) or 1.5 mol/L ethyl glycol (protocol B, group 3, n = 6). The ovarian tissue was examined with LIVE/DEAD fluorescent viability staining and histologically after isotransplantation. RESULTS: The healthy follicular loss (intact oocyte and >50% granulosa cells alive) due to cryopreservation was 15.5% with protocol A and 12.2% with protocol B. Histological examination showed follicles in all developmental phases in all groups: group 1, 35.5 +/- 5.7/mm(2) (mean +/- SD); group 2, 16.0 +/- 5.0/mm(2); group 3, 17.3 +/- 5.7/mm(2). The differences between groups 1 and 2 and between groups 1 and 3 were significant (P < 0.001). The difference between groups 2 and 3 was not significant (P = 0.33). CONCLUSIONS: These results demonstrate that the use of an open freezing system with both freezing protocols allows cryopreservation of rat ovarian tissue with equally good survival rates.