目的：探讨采用经己酮可可碱（pentoxifylline,PF）处理后的睾丸不活动精子,行卵细胞胞质内单精子注射（(intracytoplasmic sperm injection,ICSI）的临床效果。方法：选取81例梗阻性无精子症患者,女方常规超促排卵,在取卵日男方行睾丸穿刺抽吸取精术,将经体外孵育处理未获得活动精子作的样本为实验组（32例）,加入含有效成分为PF的稀少精子体外培养液20 min后,挑选出有活动力的精子进行ICSI操作;另选择穿刺可见活动精子的患者为对照组（49例）,直接进行ICSI操作。比较2组的受精率、正常受精率[2原核（2PN）率]、完全受精失败率、卵裂率、可用胚胎率、胚胎种植率、流产率、临床妊娠率等指标。结果：实验组（不动精子组）与对照组（活动精子组）间的成熟卵子百分比（80.2%比82.8%）、受精率（65.9%比65.9%）、异常受精率(7.6%比4.8%)差异无统计学意义(P>0.05)。2组间的受精失败发生率差异亦无统计学意义（3.1%比10.2%,P>0.05）,但2组间的卵裂率差异有统计学意义(91.4%比97.2%,P=0.007)。不动精子组中行非优胚胎囊胚培养的百分比明显高于活动精子组（71.9%比46.9%）,不动精子组可供移植或冷冻的D3或D5胚胎明显少于活动精子组（40.9%比52.2%,P=0.021）。2组间的临床妊娠率（39.1%比39.3%）、胚胎种植率（25.6%比28.0%）及流产率、活产率差异均无统计学意义。冷冻睾丸（附睾）精子中,不动精子组的受精率、卵裂率、可用胚胎率、临床妊娠率、胚胎种植率均有低于活动精子组的趋势,但由于样本量较小,差异尚无统计学意义（P>0.05）;而在新鲜睾丸（附睾）精子中,不动精子组的以上指标均与活动精子组相接近（P>0.05）。结论：在睾丸（附睾）不动精子标本中加入PF,使不运动的精子获得运动能力,可帮助判断并挑选出有活性的精子,达到或接近于睾丸活动精子的水平,而其安全性还需进一步验证。
Objective: The testicular sperm from biopsy and frozen/thawed tissue are frequently immotile. The purpose of this study is to assess the effect of short exposure of testicular samples with immotile sperm to pentoxifylline (PF)-a sperm motility stimulator. Method: Eight-one cases with testicular sperm ablation/testicular sperm extraction-intracytoplasmic sperm injection (TESA/TESE-ICSI) cycles in patients with azoospermia were divided into 2 groups. In group A, only immotile sperm were found in biopsies after 2 hours of incubation of tissue in culture medium, and were then treated for 20 minutes with pentoxifylline (PF) and selected the sperm with motility before ICSI(n=32). In group B, ICSI was performed with that having motile sperm found at first (n=49). Oocyte maturity, fertilization rate (2PN rate), total fertilization failure rate, embryo cleavage rate and proportion of transferable embryos as well as pregnancy rate and implantation rate were compared between the two groups. Results: Both groups had the same oocyte maturation rate（80.2% vs 82.8%）, fertilization rate（65.9% vs 65.9%）, proportion of abnormal fertilization (7.6% vs 4.8%), and the similar proportion of total fertilization failure (3.1% vs 10.2%, P>0.05）. However, when compared with that having motile sperm at first group, embryo cleavage rate was significantly lower and proportion of bastocyst culture of poor quality embryos was higher, and transfe-rable embryos was less in the PF group (91.4% vs 97.2%, P=0.007). The clinical pregnancy rate per cycle(39.1% vs 39.3%), implantation rate（25.6% vs 28.0%） and abortion rate and live birth rate were not different between the two groups. Conclusions: Treated with PF in cases of immotile testicular sperm can cause the movement of testicular sperm, allowing easier identification of vital sperm, shortening the procedure, improving the fertilization rates, lowering the chance of total fertilization failure and improving the ICSI outcome.
[1] 冯双苗, 苏迎春, 孙莹璞, 等. 附睾和睾丸精子活力对卵胞浆内单精子注射结局的影响[J]. 生殖医学杂志,2009,18(1):29-32.
[2] de Croo I, van der Elst J, Everaert K, et al. Fertilization, pregnancy and embryo implantation rates after ICSI in cases of obstructive and non-obstructive azoospermia[J]. Hum Reprod,2000,15(6):1383-1388.
[3] Nagy ZP, Joris H, Verheyen G, et al.Correlation between motility of testicular spermatozoa, testicular histology and the outcome of intracytoplasmic sperm injection[J]. Hum Reprod,1998,13(4):890-895.
[4] 陈晓宁, 张军强, 曹善仁, 等. 睾丸内不活动精子行卵细胞胞质内单精子注射结局(附3例报告)[J]. 中华男科学杂志,2008,14(12):1135-1137.
[5] de Croo I, van Coppenolle C, Everaert K, et al. ART labo-ratory: ICSI, MESA, TESE[J]. Hum Reprod, 2005,20(Suppl 1):i154-i155.
[6] 周颖, 黄学锋, 李澄棣, 等. 精子低渗肿胀实验选择完全不活动精子行ICSI获成功妊娠一例报告[J]. 中华泌尿外科杂志,2003,24(9):613.
[7] Sallam HN, Farrag A, Agameya AF, et al.The use of the modified hypo-osmotic swelling test for the selection of immotile testicular spermatozoa in patients treated with ICSI: a randomized controlled study[J]. Hum Reprod,2005,20(12):3435-3440.
[8] Aktan TM, Montag M, Duman S, et al.Use of a laser to detect viable but immotile spermatozoa[J]. Andrologia,2004,36(6):366-369.
[9] Tal J, Ziskind G, Paltieli Y, et al.ICSI outcome in patients with transient azoospermia with initially motile or immotile sperm in the ejaculate[J]. Hum Reprod,2005, 20(9):2584-2589.
[10] Kovacic B, Vlaisavljevic V, Reljic M.Clinical use of pentoxifylline for activation of immotile testicular sperm before ICSI in patients with azoospermia[J]. J Androl,2006,27(1):45-52.
[11] Palermo GD, Schlegel PN, Colombero LT, et al.Aggressive sperm immobilization prior to intracytoplasmic sperm injection with immature spermatozoa improves fertilization and pregnancy rates[J]. Hum Reprod,1996,11(5):1023-1029.
[12] 张静雯, 伍捷阳, 池霖生, 等. 睾丸不动精子行卵细胞浆内单精子注射的临床效果分析[J]. 中国优生与遗传杂志,2013,21(7):107-109.
[13] Aparicio NJ, de Turner EA, Schwarzstein L, et al. Effect of the phosphodiesterase inhibitor Pentoxyfylline on human sperm motility[J]. Andrologia,1980,12(1):49-54.
[14] Kay VJ, Coutts JR, Robertson L.Pentoxifylline stimulates hyperactivation in human spermatozoa[J]. Hum Reprod,1993,8(5):727-731.
[15] Yovich JL.Pentoxifylline: actions and applications in assisted reproduction[J]. Hum Reprod,1993,8(11):1786-1791.
[16] Huang PT, Chen SU, Chao KH, et al.Effects of fertilization promoting peptide, adenosine, and pentoxifylline on thawed human sperm[J]. Arch Androl,2003,49(2):145-153.
[17] Kay VJ, Coutts JR, Robertson L.Effects of pentoxifylline and progesterone on human sperm capacitation and acrosome reaction[J]. Hum Reprod,1994,9(12):2318-2323.
[18] Seshagiri PB, Thomas M, Sreekumar A, et al.Pentoxifylline induced signalling events during capacitation of hamster spermatozoa: significance of protein tyrosine phosphorylation[J]. Cell Mol Biol (Noisy-le-grand),2003, 49(3):371-380.
[19] Nassar A, Mahony M, Blackmore P, et al.Increase of intracellular calcium is not a cause of pentoxifylline-induced hyperactivated motility or acrosome reaction in human sperm[J]. Fertil Steril,1998,69(4):748-754.
[20] Nassar A, Morshedi M, Mahony M, et al.Pentoxifylline stimulates various sperm motion parameters and cervical mucus penetrability in patients with asthenozoospermia[J]. Andrologia,1999,31(1):9-15.
[21] Bajpai M, Doncel GF.Involvement of tyrosine kinase and cAMP-dependent kinase cross-talk in the regulation of human sperm motility[J]. Reproduction,2003,126(2):183-195.
[22] Terriou P, Hans E, Giorgetti C, et al.Pentoxifylline initiates motility in spontaneously immotile epididymal and testicular spermatozoa and allows normal fertilization, pregnancy, and birth after intracytoplasmic sperm injection[J]. J Assist Reprod Genet,2000,17(4):194-199.
[23] Griveau JF, Lobel B, Laurent MC, et al.Interest of pentoxifylline in ICSI with frozen-thawed testicular spermatozoa from patients with non-obstructive azoospermia[J]. Reprod Biomed Online,2006,12(1):14-18.
[24] 章师平, 周平, 章志国, 等. 未见原核的卵母细胞发育形成的优质胚胎的移植价值分析[J]. 安徽医科大学学报,2013,48(1):57-59.
[25] 韦多, 张翠莲, 宋小兵, 等. IVF-ET周期中1PN及0PN胚胎的非整倍体研究进展[J]. 河南医学研究,2012,21(3):374-377.
[26] Tournaye H, van der Linden M, van den Abbeel E, et al. Effects of pentoxifylline on in-vitro development of preimplantation mouse embryos[J]. Hum Reprod,1993,8(9):1475-1480.
[27] Tournaye H, van der Linden M, van den Abbeel E, et al. Mouse in vitro fertilization using sperm treated with pentoxifylline and 2-deoxyadenosine[J]. Fertil Steril,1994,62(3):644-647.
