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。     

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。     

Follicle development in grafted mouse ovaries after cryopreservation and subcutaneous transplantation

OBJECTIVE: Our aim was to determine the impact of freezing, thawing, and subcutaneous transplantation on follicular development in grafted mouse ovaries. STUDY DESIGN: The mice were divided into 3 groups: control (group 1), frozen-thawed grafting (group 2), and frozen-thawed grafting with human menopausal gonadotropin injection (group 3). After freezing and thawing, the ovaries were transplanted into the subcutaneous tissue. Two weeks after transplantation, grafted ovaries and blood samples were collected. RESULTS: Ovaries from group 3 contained significantly more follicles (246 +/- 43 follicles) than group 2(P <.05). The pattern and intensity of Cx37 immunohistochemical staining was similar in all groups. Follicle-stimulating hormone concentrations were significantly decreased in group 2 after ovarian grafting. CONCLUSION: In mice, gonadotropin treatment before subcutaneous grafting improved the survival of growing follicles. Subcutaneous ovarian transplantation may restore ovarian function and could obviate many of the problems that are related to ovarian banking for humans.     

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。     

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.     

Morphological alterations and DNA fragmentation in oocytes from primordial and primary follicles after freezing-thawing of ovarian cortex in sheep

OBJECTIVE: To evaluate DNA fragmentation in the oocyte of primordial and primary follicles and morphology of these follicles after freezing and thawing of ovarian cortex in sheep using two freezing protocols. DESIGN: Fragmentation of DNA was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) technique. SETTING: Fertility clinic in a large university hospital. ANIMALS: Five- to 6-month-old lambs. INTERVENTION(S): Two-millimeter-thick slices of hemi-ovary cortex were prepared. MAIN OUTCOME MEASURE(S): Histological structure and DNA fragmentation. RESULT(S): In the frozen fragments, the percentage of morphologically normal follicles was significantly lower for both protocols compared with the case of the control group of fresh fragments. There was no significant difference between the two types of freezing protocols (60.4% +/- 13.2% vs. 68.4% +/- 13.7%). However, the distribution of abnormalities (nucleus, cytoplasm, and nucleus and cytoplasm) was dissimilar. The results of the TUNEL technique for the three groups showed no significant difference, but the percentage of the TUNEL-positive follicles was slightly lower for the frozen fragments for both protocols with respect to the control group. CONCLUSION(S): The freezing and thawing process of the ovarian cortex does not induce fragmentation of the DNA on the oocyte of primary and primordial follicles.     

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。     

Bypassing the effect of zona pellucida changes on embryo formation following cryopreservation of bovine oocytes

OBJECTIVE: To evaluate the effects of zona pellucida changes following the cryopreservation of bovine oocytes using two different techniques. DESIGN: Prospective randomised study. SETTING: University research facility and IVF research laboratory, NURTURE, University of Nottingham, UK. INTERVENTION: 508 bovine oocytes were collected from abattoir-derived ovaries and cryopreserved by slow freezing using standard straws and a new vitrification method using our self-constructed cryoloops. After thawing, the oocytes were inseminated by ICSI and standard IVF. MAIN OUTCOME MEASURES: Oocyte degeneration and oocyte cleavage. RESULTS: Cleavage rates after IVF in fresh oocytes was higher (49.5%) than the cryopreservation groups (slow freezing: 15.4% and vitrification: 25.8%), whereas after ICSI, the rates were similar (fresh: 17.3%, slow freezing: 9.4%, vitrification: 16.0%). CONCLUSIONS: Zona pellucida changes after oocyte cryopreservation significantly reduce the embryo formation rates after IVF. ICSI bypasses this complication and in selected species it could be an appropriate method of insemination.     

3种玻璃化液对新生鼠卵巢的渗透反应及冻存效果的比较

目的:探索适宜新生鼠卵巢保存的玻璃化液和冷冻方案。方法:观察新生SD大鼠卵巢在预平衡液及3种玻璃化液中不同时间段的表面积变化,冻融后进行组织学观察和成年SD大鼠肾被膜下异体移植后在体活力分析。结果:新生鼠卵巢在预平衡液中出现渗透性脱水变化,移入EFS40(A组)、EG5.5(B组)、EG5.5/30(C组)3种玻璃化液中,再次剧烈皱缩。3min后,卵巢表面积分别为等渗液对照组表面积的63.2%、82.4%、70.8%。此脱水状态的卵巢玻璃化冻融后形态完整的卵泡百分率均与新鲜移植组(D组)无显著性差异(P>0.05);异体移植后,动情周期出现率和动情周期出现时间均与D组无显著性差异(P>0.05)。各冷冻移植组存活移植物均可见不同发育阶段的各级卵泡,但卵泡数目少于D组,移植20d时A组与D组间有显著性差异(P<0.05);移植60d时B组卵泡数少于D组,组间有差异(P<0.05);C组在各时间点上取材的卵泡数与D组均无差异(P>0.05)。结论:在预平衡液中15min、改良的EG5.5/30液中3min的二步渗透平衡法适宜新生鼠卵巢的玻璃化冷冻。     

Effect of mouse ovarian tissue cryopreservation by vitrification with Rapid-i closed system

Purpose

Currently, open systems are mainly used for cryopreservation of ovarian tissue, oocytes, and embryos, but there is a potential risk of contamination. This study was performed to assess ovarian tissue cryopreservation by a closed vitrification system (Rapid-i vitrification system?), which is already used clinically for oocyte/embryo cryopreservation.

Methods

Ovaries of C57BL/6J mice were frozen and thawed by using the Rapid-i vitrification system? (Rapid-i) followed by implantation into recipient mice. Hematoxylin-eosin staining was performed for histological examination of the frozen-thawed ovaries to assess follicle grade. Fertility after implantation of the ovaries was assessed from the live birth rate and the number of live pups.

Results

There was no significant difference in grade 1 primary follicles between fresh ovaries (control group, 94.2?±?2.9%) and frozen-thawed ovaries (Rapid-i group, 87.1?±?1.8%). However, there was a significant decrease in grade 1 early and late secondary follicles in the Rapid-i group compared with the control group. The live-birth rate was significantly lower in the Rapid-i group compared with the control group (29.2 vs. 83.3%, p?<?0.05). On the other hand, there was no significant difference in the average number of live pups between the control group and the Rapid-i group (3?±?0.4 vs. 2.7?±?0.3).

Conclusions

The Rapid-i seems to be effective for cryopreservation of mouse ovarian tissue. Under appropriate conditions, the Rapid-i could be employed for ovarian tissue cryopreservation and preservation of fertility in humans.

     

Methods for cryopreservation of human ovarian tissue

Human ovarian tissue can be successfully cryopreserved, with good survival and function after thawing. Experimental animal studies regarding ovarian tissue cryopreservation resulting in live-born offspring preceded the present freezing systems in humans. On the basis of current knowledge, the standard method for human ovarian cryopreservation is slow programmed freezing, using human serum albumin-containing medium, and propanediol, dimethylsulphoxide (DMSO) or ethylene glycol as a cryoprotectant, combined with sucrose. Vitrification is still at the experimental stage. Whole organ cryopreservation is an interesting experimental option. Transplantation of the frozen-thawed tissue is a feasible method to utilize the tissue in infertility treatment. Ovarian function has been restored in humans. Because one healthy child has already been born from cryopreserved tissue, tissue cryopreservation should perhaps be offered to all young girls and women who can be predicted to undergo premature ovarian failure due to cancer treatment or genetic causes. Maturation of follicles in vitro from frozen-thawed tissue is another option that is still under development.     

Cryopreservation of human ovarian tissue by solid-surface vitrification

OBJECTIVE: To cryopreserve human ovarian tissue using solid-surface vitrification (SSV) technique for the first time. STUDY DESIGN: Human ovarian slices from each of 26 patients were randomly allocated to fresh, SSV, and slow-freezing groups, respectively. Histological observation and the TUNEL assay of the tissue were performed after cryopreservation. In vitro culture was done to study the initial recruitment of follicles and hormone production ability after SSV/slow-freezing. RESULTS: The majority of primordial follicles were maintained intact through either SSV or the slow-freezing method. No statistically significant destructive effect of SSV or slow-freezing for primordial follicles and stromal cells was found using the TUNEL assay. In the SSV and slow-freezing groups, estradiol and progesterone were secreted continuously during 10 days in culture, and the proportions of growing follicles increased significantly comparing to the uncultured fresh group. The follicular proportions and the concentrations of estradiol and progesterone exhibited no statistically significant differences between the SSV and slow-freezing groups. CONCLUSION: SSV is an effective, simple and inexpensive alternative for human ovarian tissue cryopreservation.     

Ovarian tissue vitrification: Cortex and whole ovary in sheep

OBJECTIVE: The aim of this study was to evaluate a cryopreservation technique by vitrification of cortex or whole ovaries in sheep, using two cryoprotectant solutions: VS1 and VS4 and to study their physical properties to avoid ice crystallisation by vitrification of whole sheep ovaries permeated with a cryoprotectant solution. ANIMALS AND METHODS: From 6-month-old ewes, whole sheep ovaries with their vascular pedicles were collected at the slaughterhouse or at the veterinary school and prepared for cryoprotectant toxicity tests and freezing procedure. Follicle viability was measured by trypan blue test and histological examination of ovary. The hemi-ovarian cortex was stored in liquid nitrogen. Four to six weeks after the first laparotomy, the controlateral ovary was removed and the vitrified-warmed hemi-ovary was sutured. Thermal properties of a cryoprotectant solution called VS4 (critical cooling rates [Vccr], vitreous transition temperature [Tg], end of melting temperature [Tm]) were measured by differential scanning calorimetry. RESULTS: No significant difference in follicle viability or normal follicle rates was observed between ovarian cortex exposed or non-exposed to cryoprotectant solutions. Nor was any significant difference observed before and after vitrification. Three pregnancies occurred, from which four lambs were born after autografts of vitrified ovarian cortex. With whole ovary, the decrease in the number of normal follicles was lower when frozen-thawed ovaries were treated with VS4 (P = 0.04). There were less nuclear anomalies (P = 0.02). The Vccr of VS4 has been estimated to be 14.3+/-1.1 degrees C/min and Tg was -125.0+/-0.2 degrees C. Because the penetration of cryoprotectants was very low, Vccr was very high and the cooling speed did not allow cortex to vitrify. DISCUSSION AND CONCLUSIONS: Cryopreservation of cortex or whole ovary by vitrification seems a promising technique in reproductive medicine. The best histologic results were obtained with the VS4 cryoprotectant when whole ovary was vitrified.     

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.     

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.     

Beneficial effect of desialylated erythropoietin administration on the frozen-thawed canine ovarian xenotransplantation

Purpose

The main drawback of ovarian cryopreservation followed by transplantation is that a large proportion of follicles are lost after transplantation. Thus, effects of erythropoietin (EPO) and desialylated EPO administration on the frozen-thawed canine ovarian xenotransplantation were examined.

Methods

The protective and survival-promoting effects of EPO and desialylated EPO on the follicles of frozen-thawed canine ovaries after transplantation were examined using NOD-SCID mice. Frozen-thawed dog ovarian tissue with 400 U/kg of EPO or asialo EPO was placed into the ovarian bursa.

Results

At 4 weeks after the transplantation, the ovaries were removed and subjected to histological examination. The survival rate of early primary follicles was 15.2% in the EPO group and 157.6% in the asialo EPO group, in contrast to 10.1% in the untreated group.

Conclusions

These results demonstrate that administration of asialo EPO could be effectively used to enhance the survival of the follicles of transplanted cryopreserved ovaries.     

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.     

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.

     

A 60-month non-comparative study on bleeding profiles with the levonorgestrel intrauterine system from the late transition period to estrogen supplemented menopause

Objective

To investigates the effect of sphingosine-1-phosphate (S1P) supplementation on follicular integrity and apoptosis in vitrified-warmed mouse ovarian grafts.

Study design

Ovaries from 4-week-aged ICR mice were vitrified using a vitrification solution with or without 2 μM S1P. After warming, follicular normality was assessed by histological analysis and TUNEL assay. A part of ovaries vitrified with or without 2 μM S1P was transplanted, and 2 weeks later, gross and microscopic follicular morphology was assessed.

Results

During vitrification and warming, inclusion of 2 μM S1P into the vitrification solution significantly raised the rate of morphologically intact follicles compared to controls (36.6% vs. 30.8%, p = 0.047). This protective effect was profound especially in primordial follicles (45.5% vs. 34.6%, p = 0.034). After transplantation of vitrified-warmed ovaries, the morphological integrity of primordial follicles was superior in the S1P-treated group (55.0% vs. 39.4%, p = 0.035). The rates of non-apoptotic follicles (TUNEL-negative) were similar in the two groups in either non-transplanted or transplanted ovaries.

Conclusion

Inclusion of S1P in the vitrification solution during transplantation of vitrified-warmed ovary had a beneficial effect on preservation of the primordial follicular pool.     

Effect of vitrification method on survivability, follicular growth and ovulation of preantral follicles in mice

AIM: This study was designed to compare the survival rates, follicular growth rates, and ovulation rates of vitrified preantral follicles (PF) from ovaries with those isolated from a vitrified ovarian cortical strip. METHODS: Mouse ovaries were divided into three groups: those not treated by vitrification of the PF (control), those treated by vitrification of the PF isolated from the ovaries (group I), and those treated by vitrification of ovarian tissue followed by PF isolation (group II). The group I samples were exposed to equilibration solution (EG-20) for 5.0 min plus vitrification solution (EFS-40) for 0.5 min, while the group II samples were exposed to EG-20 for 10.0 min plus EFS-40 for 2.0 min, before vitrification. They were subsequently placed on an electron microscope grid, and submerged immediately in liquid nitrogen. After thawing, the survival rate and the growth rate of the follicles were evaluated every 2 days. RESULTS: In the in vitro condition, the follicles grew and developed into antral follicles in groups I and II. The survival rate of the group I samples was higher than that of the group II samples during the in vitro culture (P<0.05). The growth rates of the follicles in group I were higher than those in group II after day 6 (P<0.05). The ovulation rate of the samples in group I was higher than that of group II (P<0.05). CONCLUSION: These results demonstrated that direct PF vitrification appeared to be better than vitrification of the PF isolated from ovarian tissue.