Behr B, Pool T B, Milki A A, Moore D, Gebhardt J, Dasig D
Department GYN/OB Stanford University Medical Center, CA 94305, USA.
Hum Reprod. 1999 Feb;14(2):454-7. doi: 10.1093/humrep/14.2.454.
This preliminary analysis was designed to quantify blastocyst development of supernumerary embryos without the use of feeder cells, conditioned medium or whole serum. Embryos derived from in-vitro fertilization (IVF) that were not transferred or cryopreserved were included in this study. Ova were harvested for IVF after a standard ovarian stimulation with gonadotrophin-releasing hormone agonist/ human menopausal gonadotrophin (GnRHa/HMG) or follicle-stimulating hormone (FSH). Ova were collected and culture in 150 microliters droplets of P1 medium under mineral oil, in groups at 37 degrees C under 5% CO2, 5% O2, 90% N2 (group A) or under 5% CO2 in air (group B) environment. Embryo transfer was performed 72 h post-harvest. Viable embryos not transferred or cryopreserved were placed in blastocyst medium and cultured for an additional 48 h in 5% CO2 in air. Embryos that exhibited an expanded blastocoelic cavity and well-defined inner cell mass at 120 h were counted. Of 838 supernumerary embryos cultured, 448 (53.5%) reached the expanded blastocyst stage by 120 h of culture. Patients were given the option of cryopreservation at that time. The embryos were cryopreserved using a standard protocol with serial addition of glycerol. Embryos reaching the blastocyst stage after more than 120 h of culture were not included. There was no difference in the proportions of blastocyst development between group A, 217/410 (53.5%) and group B, 231/428 (54%). To date, 16 patients have each had up to three thawed blastocysts transferred, out of whom seven became pregnant. This report demonstrates that a simple system of sequential culture generated acceptable, viable blastocyst development (54%) with supernumerary embryos, without the use of feeder cells, conditioned medium or whole serum. Recognizing the differential metabolic requirements of early and late cleavage stage embryos has enabled the application of a glucose/phosphate-free simple culture medium (P1) for up to 72 h of culture and a complex, glucose-containing medium (blastocyst medium) for subsequent blastocyst development.
本初步分析旨在量化多余胚胎的囊胚发育情况，且不使用饲养细胞、条件培养基或全血清。本研究纳入了体外受精（IVF）获得的未移植或未冷冻保存的胚胎。在使用促性腺激素释放激素激动剂/人绝经期促性腺激素（GnRHa/HMG）或促卵泡激素（FSH）进行标准卵巢刺激后，采集卵子用于IVF。卵子收集后，在矿物油下的150微升P1培养基液滴中培养，在37℃、5%二氧化碳、5%氧气、90%氮气环境下分组培养（A组），或在空气中5%二氧化碳环境下培养（B组）。收获后72小时进行胚胎移植。未移植或未冷冻保存的存活胚胎置于囊胚培养基中，在空气中5%二氧化碳环境下再培养48小时。对在120小时时呈现扩张囊胚腔和明确内细胞团的胚胎进行计数。在培养的838个多余胚胎中，448个（53.5%）在培养120小时时达到扩张囊胚阶段。此时患者可选择冷冻保存。胚胎使用添加甘油的标准方案进行冷冻保存。培养超过120小时后达到囊胚阶段的胚胎未纳入。A组（217/410，53.5%）和B组（231/428，54%）的囊胚发育比例无差异。迄今为止，16名患者每人移植了多达3个解冻囊胚，其中7人怀孕。本报告表明，一个简单的序贯培养系统能够使多余胚胎产生可接受的、有活力的囊胚发育（54%），且不使用饲养细胞、条件培养基或全血清。认识到早期和晚期卵裂期胚胎不同的代谢需求，使得能够应用不含葡萄糖/磷酸盐的简单培养基（P1）进行长达72小时的培养，并应用复杂的含葡萄糖培养基（囊胚培养基）进行后续的囊胚发育。
