Machlin J H, Hannum D F, Jones A S K, Schissel T, Potocsky K, Marsh E E, Hammoud S, Padmanabhan V, Li J Z, Shikanov A
Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, MI, USA.
Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA.
Hum Reprod. 2025 Apr 1;40(4):683-694. doi: 10.1093/humrep/deaf009.
Does the slow-freezing and thawing process have a negative impact on the transcriptome of oocytes isolated from early-stage human follicles compared to fresh controls?
The transcriptional profiles of fresh and frozen/thawed oocytes did not cluster separately, indicating undetectable differences between the two groups when compared to within-donor heterogeneity.
Previous studies using histological analysis of follicle morphology, density, and stage distribution in slow-frozen/thawed human ovarian cortex compared to fresh controls showed no differences between the two groups. Clinical cases reported in the past 10 years have demonstrated that transplanted slow-frozen/thawed and fresh ovarian cortex restored normal serum FSH levels and regular menstrual cycles by 5 months. However, the slow-frozen and thawed tissue resulted in lower rates of pregnancies and live births, albeit not statistically significant.
STUDY DESIGN, SIZE, DURATION: We utilized single-cell RNA-sequencing (scRNAseq) of 144 human oocytes isolated from cadaver ovaries obtained from three donors.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Human ovarian cortex from three healthy premenopausal donors 16, 18, and 27 years old was cut into squares measuring 10 × 10 × 1 mm3 and either slow-frozen and thawed or processed fresh. First, using a novel method for isolating live oocytes from primordial and primary follicles, the ovarian cortex squares were fragmented with a McIlwain tissue chopper and enzymatically digested. Next, oocytes were mechanically denuded under a dissection microscope and placed individually into wells containing lysis buffer for scRNAseq. Lysed single oocytes were subjected to library prep using the seqWell PlexWell rapid single-cell RNA protocol. Pooled libraries were subjected to 150-bp paired-end sequencing on the NovaSeq6000 Illumina platform. In total, we sequenced 144 oocytes-24 oocytes isolated fresh and 24 oocytes isolated after slow-freezing and thawing from each of the three donors. Additionally, we performed histological analysis of fresh and frozen/thawed ovarian cortex tissue from all three donors using hematoxylin and eosin staining and analyzed morphology, follicle density, and follicle stage distribution differences between fresh and cryopreserved ovarian cortex.
The histological analysis revealed no differences in follicle stage distribution or follicle morphology between conditions, with the percentage of normal follicles in fresh and frozen/thawed tissue, respectively, as 86.7% and 91.0% for Donor 1, 91.7% and 92.5% for Donor 2, and 96.1% and 91.1% for Donor 3. The follicle density per mm3 in fresh and frozen/thawed tissue, respectively, was 279.4 and 235.8 for Donor 1, 662.2 and 553.5 for Donor 2, and 55.8 and 71.4 for Donor 3. The difference in follicle density was not statistically significant between fresh and frozen/thawed conditions for Donors 2 and 3, and significant (P = 0.017) for Donor 1. The stromal cell densities in fresh and frozen/thawed tissue, respectively, were 0.014 in both conditions for Donor 1, 0.014 and 0.016 for Donor 2, and 0.013 and 0.014 for Donor 3. There was no statistically significant difference in stromal cell density between conditions in Donor 1 and Donor 3, though it was statistically significant (P ≤ 0.001) for Donor 2. The transcriptional profiles of fresh and frozen/thawed oocytes did not cluster separately, suggesting insignificant differences between the two groups. However, at the group mean level, there was a small shift between the fresh and frozen/thawed oocytes and the shifts were parallel across the three donors. In this comparison, fresh oocytes were enriched for gene ontology terms related to chromosome segregation and mitosis, whereas frozen/thawed oocytes were enriched for terms related to wound response, cAMP signaling, and extracellular matrix organization.
Datasets available on Zenodo.org. DOI: https://zenodo.org/records/13224872.
LIMITATIONS, REASONS FOR CAUTION: In this study, we only sequenced the oocytes isolated from early-stage follicles due to technical challenges collecting and sequencing the somatic cells surrounding the oocytes. Investigating the transcriptomic changes after freezing and thawing in the somatic cells would need to be studied in the future. Additionally, we built RNAseq libraries immediately after thawing focusing on the immediate changes. Investigation of the effects that manifest at later timepoints, either in culture or upon implantation in an animal model, may reveal additional effects of the freeze/thaw process on the transcriptome.
The only clinically approved method of fertility preservation for prepubertal cancer patients and adult patients who cannot delay cancer treatment is ovarian tissue cryopreservation. Investigation of cryopreservation-induced changes in follicles at all stages is critical to further our understanding of the safety and efficacy of using these tissues for fertility preservation in the clinic. Our study is the first to analyze transcriptomic changes between individual fresh and slow-frozen/thawed human oocytes collected from early-stage follicles. To accomplish this, we developed a novel method for dissociating both fresh and frozen/thawed human ovarian cortex to obtain live denuded oocytes from early-stage follicles. Our findings provide insights into the use of cryopreserved tissue and follicles for fertility preservation efforts.
STUDY FUNDING/COMPETING INTEREST(S): This work was funded by National Institutes of Health (NIH) R01HD099402, Career Training in Reproductive Biology (CTRB) Training Grant National Institutes of Health (NIH) T32 to Jordan Machlin, National Institutes of Health (NIH) F31-HD106626 and National Institutes of Health (NIH) T31H-D079342 to Andrea Jones, National Institutes of Health (NIH) T32-GM70449 to D. Ford Hannum, and The Chan Zuckerberg Initiative Grant CZF2019-002428. We have no conflicts of interest to declare.
与新鲜对照组相比，慢速冷冻和解冻过程是否会对从早期人类卵泡中分离出的卵母细胞转录组产生负面影响？
新鲜和冷冻/解冻卵母细胞的转录谱没有分开聚类，表明与供体内的异质性相比，两组之间没有可检测到的差异。
先前的研究通过对慢速冷冻/解冻的人类卵巢皮质与新鲜对照组进行卵泡形态、密度和阶段分布的组织学分析，结果显示两组之间没有差异。过去10年报告的临床病例表明，移植的慢速冷冻/解冻和新鲜卵巢皮质在5个月内恢复了正常的血清促卵泡激素水平和规律的月经周期。然而，慢速冷冻和解冻的组织导致怀孕率和活产率较低，尽管没有统计学意义。
研究设计、规模、持续时间：我们对从三名供体的尸体卵巢中分离出的144个人类卵母细胞进行了单细胞RNA测序（scRNAseq）。
参与者/材料、设置、方法：将来自三名年龄分别为16岁、18岁和27岁的健康绝经前供体的人类卵巢皮质切成10×10×1立方毫米的方块，然后进行慢速冷冻和解冻或新鲜处理。首先，使用一种从原始卵泡和初级卵泡中分离活卵母细胞的新方法，用麦基尔温组织切碎机将卵巢皮质方块切碎并进行酶消化。接下来，在解剖显微镜下机械去除卵母细胞周围的颗粒细胞，并将其单独放入含有裂解缓冲液的孔中进行scRNAseq。裂解的单个卵母细胞使用seqWell PlexWell快速单细胞RNA方案进行文库制备。合并后的文库在Illumina NovaSeq6000平台上进行150碱基对的双端测序。我们总共对144个卵母细胞进行了测序——从三名供体中的每一名中新鲜分离出24个卵母细胞，慢速冷冻和解冻后分离出24个卵母细胞。此外，我们使用苏木精和伊红染色对所有三名供体的新鲜和冷冻/解冻卵巢皮质组织进行了组织学分析，并分析了新鲜和冷冻保存的卵巢皮质之间的形态、卵泡密度和卵泡阶段分布差异。
组织学分析显示，不同条件下卵泡阶段分布或卵泡形态没有差异，供体1新鲜组织和冷冻/解冻组织中正常卵泡的百分比分别为86.7%和91.0%，供体2为91.7%和92.5%，供体3为96.1%和91.1%。供体1新鲜组织和冷冻/解冻组织中每立方毫米的卵泡密度分别为279.4和235.8，供体2为662.2和553.5，供体3为55.8和71.4。供体2和供体3新鲜和冷冻/解冻条件下的卵泡密度差异无统计学意义，供体1差异有统计学意义（P = 0.017）。供体1新鲜和冷冻/解冻组织中的基质细胞密度在两种条件下均为0.014，供体2为0.014和0.016，供体3为0.013和0.014。供体1和供体3不同条件下的基质细胞密度无统计学差异，尽管供体2差异有统计学意义（P≤0.001）。新鲜和冷冻/解冻卵母细胞的转录谱没有分开聚类，表明两组之间差异不显著。然而，在组平均水平上，新鲜和冷冻/解冻卵母细胞之间有一个小的偏移，并且在三名供体中偏移是平行的。在这种比较中，新鲜卵母细胞在与染色体分离和有丝分裂相关的基因本体术语上富集，而冷冻/解冻卵母细胞在与伤口反应、cAMP信号传导和细胞外基质组织相关的术语上富集。
可在Zenodo.org上获取数据集。DOI：https://zenodo.org/records/13224872。
局限性、谨慎原因：在本研究中，由于在收集和测序卵母细胞周围的体细胞时存在技术挑战，我们仅对从早期卵泡中分离出的卵母细胞进行了测序。未来需要研究冷冻和解冻后体细胞的转录组变化。此外，我们在解冻后立即构建RNAseq文库，重点关注即时变化。研究在培养或植入动物模型中稍后时间点出现的影响，可能会揭示冷冻/解冻过程对转录组的额外影响。
对于青春期前癌症患者和不能延迟癌症治疗的成年患者，唯一临床批准的生育力保存方法是卵巢组织冷冻保存。研究冷冻保存对各个阶段卵泡的影响对于进一步了解在临床中使用这些组织进行生育力保存的安全性和有效性至关重要。我们的研究是首次分析从早期卵泡中收集的单个新鲜和慢速冷冻/解冻人类卵母细胞之间的转录组变化。为了实现这一点，我们开发了一种新方法来解离新鲜和冷冻/解冻的人类卵巢皮质，以从早期卵泡中获得活的裸卵母细胞。我们的研究结果为使用冷冻保存的组织和卵泡进行生育力保存提供了见解。
研究资金/利益冲突：这项工作由美国国立卫生研究院（NIH）R01HD099402资助，生殖生物学职业培训（CTRB）培训资助美国国立卫生研究院（NIH）T32授予乔丹·马克林，美国国立卫生研究院（NIH）F31-HD106626和美国国立卫生研究院（NIH）T31H-D079342授予安德里亚·琼斯，美国国立卫生研究院（NIH）T32-GM70449授予D. 福特·汉纳姆，以及陈·扎克伯格倡议资助CZF2019-002428。我们没有利益冲突需要声明。
