Vitrified Wharton’s jelly tissue as a biomaterial for multiple tissue engineering applications

Abstract

Wharton’s Jelly (WJ) tissue is a promising biomaterial, for tissue engineering applications. However, its preservation over a long period in order to be readily available needs further optimization. A possible solution could be the vitrification and storage of WJ tissue at low temperatures. The aim of this study is to evaluate the effect of low temperature in the WJ tissue, which was stored at ?196?°C, either with the vitrification or conventional cryopreservation methods. WJ tissues were isolated from human umbilical cords, cryopreserved with the above methods and remained for 1?year at ?196?°C. Histological analysis of tissue’s extracellular matrix (ECM), isolation, and characterization of mesenchymal stromal cells (MSCs) were performed. Histological analysis revealed the presence of ECM components such as collagen, sulfated glycosaminoglycans (sGAGs), and the presence of cell nuclei only in vitrified samples. Furthermore, MSCs were isolated and expanded successfully from vitrified WJ tissues, whereas a few viable cells were obtained from conventionally cryopreserved tissues that were not further expanded. In conclusion, this study indicated the proper preservation of vitrified WJ tissues after 1?year of storage, which eventually could be used in tissue engineering and regenerative medicine approaches.     

Maturation in vitro des ovocytes en préservation de la fertilité féminine

Recovering immature oocytes from unstimulated ovaries, followed by in vitro maturation (IVM) was initially proposed to avoid the risks and side effects of exogenous gonadotropin administration. Therefore, during the past decades, IVM was mainly offered to patients with polycystic ovary syndrome at high risk of ovarian hyperstimulation syndrome. However, the development of fertility preservation has recently opened new perspectives in the field of IVM. The present review reports the possible indications of IVM, in the strategy of female fertility preservation.     

囊胚玻璃化冷冻保存时长对复苏移植临床结局的影响

目的研究使用开放式载体玻璃化冷冻保存囊胚的时长对复苏移植妊娠结局和新生儿结局的影响,探讨胚胎经过玻璃化冷冻并进行6年以上的长期保存是否对临床结局产生负面影响。方法回顾性分析2006年3月至2018年12月期间在我院生殖中心进行囊胚玻璃化冷冻复苏移植的2 643例患者的临床资料。按照胚胎玻璃化冷冻保存时长将其分为7组:≤1年,1~2年,2~3年,3~4年,4~5年,5~6年和≥6年,比较各组患者一般情况、临床结局、活产情况和单胎新生儿出生结局之间的差异,再使用二分类Logistic回归分析女性取卵年龄、移植年龄、胚胎冷冻保存时间、囊胚冷冻日期和囊胚级别对妊娠率和活产率的影响。结果各组患者取卵年龄、移植年龄、优胚率、复苏胚胎存活率、妊娠率和活产率组间差异有统计学意义(P<0.05)。各组平均移植胚胎数、D5胚胎比例、种植率、流产率、异位妊娠率、单胎率、出生男孩率、出生缺陷率、单胎出生体重、身长和孕天数均无统计学差异(P>0.05)。Logistic回归分析显示取卵年龄、移植年龄和冷冻保存时间对妊娠率无显著影响(P>0.05),取卵年龄和冷冻保存时长对活产率无显著影响(P>0.05),而增加移植D5胚胎和优质胚胎数量可以提高妊娠率和活产率(P<0.05),移植年龄的增加会降低活产率(P<0.05)。结论使用开放性载体玻璃化冷冻保存囊胚的时长对复苏后移植的妊娠结局和新生儿结局没有明显影响。囊胚经过玻璃化冷冻并进行6年以上长期冷冻保存,临床结局未产生明显负面影响。     

Assessment of vitrification outcome by xenotransplantation of ovarian cortex pieces in γ-irradiated mice: morphological and molecular analyses of apoptosis

Purpose

The aim of this study was the investigation of caspase-3/7 activity and apoptosis related gene expression after vitrification and xenotransplantation of human ovarian fragments.

Methods

Ovarian specimens were obtained from normal female-to-male transsexual women during laparoscopic surgery and cut into small pieces and were considered as vitrified and non-vitrified groups. The morphological study, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, caspase-3/7 activity and apoptosis related gene expression analysis were done in both non-vitrified and vitrified groups in two steps (before transplantation of ovarian tissues and 30 days after transplantation).

Result(s)

In spite of high rate of normal follicles in both non-transplanted tissues these rates were significantly decreased in vitrified and non-vitrified grafted tissues, moreover grafted-vitrified tissue showed significantly less normal follicles than grafted-non-vitrified group (P < 0.05). The expression of some pro and anti-apoptotic genes in vitrified-warmed tissues were not changed compared to non-vitrified ones but the expression of Fas and caspase8 was increased and the expression of BRIC5 was decreased in this group (P < 0.05). In transplanted vitrified group the Bcl2, FasL and BRIC5 gene expression was high and caspase8 was low (P < 0.05). The expression of all genes in both grafted groups was more than non-grafted tissues except for caspase8 (P < 0.05). The TUNEL positive signals and caspase-3/7 activity were increased in both grafted groups compared to non-grafted groups and this enzyme activity in grafted-vitrified group was more than grafted-non-vitrified group (P < 0.05).

Conclusion(s)

This study provides the first evidence on the significant effect of vitrification on follicular apoptosis of grafted human ovarian tissue at mRNA level. The signs of follicular survival or degeneration detected by morphological assessment and caspase-3/7 activity were closely correlated to the changes in expression of apoptosis-related genes.     

大鼠卵巢组织冷冻复苏及自体异位移植研究

目的：探讨直接覆盖玻璃化冷冻(direct cover vitrification, DCV)在冷冻大鼠卵巢组织中的效果,并对自体异位移植后卵巢组织存活状况及内分泌情况进行分析。方法性成熟的 Wistar 大鼠50只随机分为 A、B、C、D 组, A 和 B 组各20只,C 和 D 组各5只。 A 组为新鲜卵巢组织移植;B 组卵巢组织经 DCV 冷冻保存2周复苏后移植;C 组为去势对照;D 组为假手术对照。自体异位移植6周后检测大鼠血清中雌二醇(E2)水平,计算卵泡密度,观察移植卵巢组织形态学及增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)的表达情况。结果大鼠卵巢组织移植存活的卵巢组织周围可见血管供应,移植失败的卵巢组织呈纤维化状态,与周围组织血供不明显。 B 组卵巢组织移植存活率低于 A 组(P <0．05)。 B 组卵巢组织中卵泡密度均低于 A、D 组,且 A 组低于 D 组(P <0．05)。 A、B 组血清 E2水平高于 C 组,低于 D 组(P <0．05)。 A、B 组卵巢组织 PCNA 阳性率均低于 D 组,且 B 组低于 A 组(P <0．05)。结论大鼠卵巢组织经 DCV 法冻融自体异位移植后部分卵泡能存活并可恢复内分泌功能。     

Aspects cliniques pratiques de la vitrification ovocytaire en oncofertilité

Oocyte vitrification is a preservation fertility strategy, which can be performed in women after puberty to preserve gametes before beginning a gonadotoxic anticancer treatment. Based on available literature and our personal data, we aim to provide an overview about the feasibility, the clinical and logistic difficulties of oocyte vitrification in the field of oncofertility: limit age for oocyte cryopreservation, time required and protocols for ovarian controlled stimulation, ovarian response to stimulation, for what hopes of pregnancy?     

三种玻璃化液对新生大鼠卵巢玻璃化冷冻效果的比较

目的 比较三种玻璃化液低温冻存新生大鼠卵巢的效果,为人卵巢组织冷冻的保护剂选择提供实验依据.方法 将36只出生1d的大鼠卵巢进行玻璃化冷冻,卵巢标本随机分为4组,新鲜对照组和EFS40、EG5.5、改良的EG5.5/30液冷冻实验组.分别通过光镜组织结构观察、荧光活力检测、TUNEL实验和免疫组织化学分析进行冷冻效果的比较.结果 EFS40、EG5.5、改良的EG5.5/30组冻融后存活卵泡的百分率低于新鲜对照组(P＜0.05);三种玻璃化液冷冻实验组卵泡凋亡率均高于新鲜对照组(P＜0.05);EFS40和EG5.5组,卵泡内Bcl-2阳性表达率较新鲜对照组下降(P＜0.05),Bax阳性表达率则较新鲜对照组上升(P＜0.05);改良的EG5.5/30组Bcl-2、Bax阳性表达率趋势与EFS40、EG5.5组相同,但均与新鲜对照组比较差异无统计学意义(P＞0.05).结论 与EFS40、EG5.5液相比,改良的EG5.5/30液更适合新生大鼠卵巢的玻璃化冻存.     

Heterotopic autotransplantation of vitrified mouse ovary

Purpose  

The aim of this study was to investigate the survival and development of premature follicles and oocytes from a vitrified-transplanted ovary in a murine experimental model.     

Comparison of two different media for vitrification and rewarming of human zygotes: Prospective randomized study

Vitrification has been successfully used for cryopreservation of human oocytes, zygotes and embryos. There are various media which contain different combinations and concentrations of cryoprotectants for this technique. In this prospective randomized study, two different vitrification and warming media were compared in human zygotes’ cryopreservation.