Extracellular vesicles (EVs) are highly specific and multi-purpose vesicular structures that are released by various cell and tissue types in the body. However, the secretion of EVs from mammalian embryos, especially human, has not been well characterized. Thus, the aim of this study was to 1) identify EVs in human preimplantation embryos at different stages of their development using scanning and electron microscopy, and 2) investigate whether EVs can cross the zona pellucida (ZP) and be released from human embryos cultured in vitro. Human oocytes, zygotes, cleavage embryos and blastocysts donated for research were labeled with the tetraspanin EV marker CD9 and analyzed by scanning and transmission electron microscopy. Embryo culture conditioned media collected 3- and 5-days post fertilization were examined for the presence of EVs using electron microscopy. We detected numerous CD9 positive vesicles released from all embryos examined. They were observed on the surface of the plasma membrane, within the perivitelline space as well as throughout the zona pellucida. Interestingly, EVs were not seen in the ZP of all mature metaphase II oocytes, however, were detected just after fertilization in the ZP of zygotes and embryos. Electron microscopy using negative staining, and nanoparticle tracking analysis (NTA) of embryo conditioned culture media also showed the presence of vesicles of various sizes, which were round shaped, and had a lipid bilayer. Their size ranged from 30 to 500 nm, consistent with the sizes of exosomes and microvesicles. In conclusion, the results of the study provide evidence that human preimplantation embryos at all developmental stages secrete EVs into the perivitelline space, which then traverse through the ZP, and are then released into the surrounding culture medium.

Abbreviations: EVs: extracellular vesicles; ZP: zona pellucida; CD9, CD63, and CD81: tetraspanin EV markers; NTA: nanoparticle tracking analysis; ESCRT: endosomal sorting complexes required for transport; SEM: scanning electron microscopy; TEM: transmission electron microscopy; TE: trophectoderm; ICM: inner cell mass; PVS: perivitelline space; MI: metaphase I; MII: metaphase II; GV: germinal vesicle; MVs/EXs: microvesicles/exosomes; hCG: human chorionic gonadotrophin; GnRH: gonadogrophin releasing hormone; ICSI: intracytoplasmic sperm injection; SPS: serum protein substitute; 1PN: one pronuclear zygote; 3PN: tri-pronuclear zygote; IgG: immunoglobulin G; PBS: phosphate buffer saline; ETHO: ethanol; ESED: Environmental Secondary Electron Detector; BSA: bovine serum albumin

细胞外囊泡（EVs）是高度特异性的多用途囊泡结构，可通过体内各种细胞和组织类型释放。然而，尚未充分表征哺乳动物胚胎，特别是人类胚胎中EV的分泌。因此，本研究的目的是：1）使用扫描和电子显微镜在人类胚胎植入前胚胎的不同发育阶段鉴定其EV，以及2）研究EV是否可以穿过透明带（ZP）并从培养的人类胚胎中释放出来。体外。捐赠给研究的人卵母细胞，受精卵，卵裂卵和囊胚用四跨膜蛋白EV标记CD9标记，并通过扫描和透射电子显微镜进行分析。受精后3天和5天收集的胚胎培养条件培养基使用电子显微镜检查EV的存在。我们检测到从所有检查的胚胎中释放出的大量CD9阳性囊泡。在质膜表面，周玻璃空间以及整个透明带中都观察到它们。有趣的是，在所有成熟的中期II卵母细胞的ZP中都没有看到EV，但是在受精卵和胚胎的ZP中受精后才发现了EV。使用负染色的电子显微镜，胚胎条件培养基的纳米颗粒追踪分析（NTA）和纳米颗粒追踪分析（NTA）也显示存在各种大小的囊泡，囊泡呈圆形，并具有脂质双层。它们的大小在30至500 nm之间，与外泌体和微泡的大小一致。总之，该研究结果提供了证据，表明人类在各个发育阶段的胚胎植入前胚胎都会将EV分泌到卵周膜空间中，然后穿过ZP，然后释放到周围的培养基中。

缩写： EVs：细胞外囊泡；ZP：透明带；CD9，CD63和CD81：四跨膜蛋白EV标记；NTA：纳米颗粒跟踪分析；ESCRT：运输所需的内体分选复合物；SEM：扫描电子显微镜；TEM：透射电子显微镜；TE：滋养外胚层；ICM：内部细胞团；PVS：玻璃体周间隙；MI：中期I；MII：中期II；GV：生发囊泡；MV / EX：微囊泡/外泌体；hCG：人绒毛膜促性腺激素；GnRH：性腺激素释放激素；ICSI：胞浆内精子注射；SPS：血清蛋白替代品；1PN：一种原核合子；3PN：三原核合子；IgG：免疫球蛋白G；PBS：磷酸盐缓冲液；ETHO：乙醇；ESED：环境二次电子检测器；BSA：牛血清白蛋白