在IVF中,血清高胎球蛋白B含量与受精率相关

Eileen Dietzel 1, JosefNeulen 2, Benjamin Rosing 2,Ute Weissenborn 2, and Willi Jahnen-Dechent 1,*

 

STUDY QUESTION  Is serum fetuin-B associated with the fertilization rate in invitro fertilization (IVF)?

SUMMARY ANSWER  Serum fetuin-B increased during IVF cycles when oocytes could befertilized while remained unchanged in fertilization failure.

WHAT IS KNOWN ALREADY  Fetuin-Bdeficiency in mice causes premature zona pellucida hardening mediated by thezona protease ovastacin. Thus fetuin-B deficiency renders females infertile.

STUDYDESIGN, SIZE, DURATION  Wedetermined the human serum fetuin-B reference range, studying longitudinally,over the course of one month, five male and seven female volunteers withouthormone treatment and four female volunteers on varying hormonal contraception.We sampled blood and determined serum fetuin-B, luteinizing hormone (LH),estradiol (E2) and progesterone (P4). In addition, we determined serum fetuin-Band estradiol in eight women undergoing intracytoplasmatic sperm injection(ICSI, nine ICSI cycles) and 19 women undergoing IVF (21 IVF cycles) afterovarian stimulation with recombinant human follicular stimulating hormone(rFSH) and/or a combined medication of FSH and LH. At least three blood sampleswere analyzed in each cycle. We compared serum fetuin-B and follicular fluidfetuin-B in nine patients by measuring follicular fetuin-B in pooled follicularfluid, and in fluid obtained from individual follicles. Samples were drawn fromJanuary 2012 to March 2014.

PARTICIPANTS/MATERIALS,SETTING, METHOD  Allvolunteers and patients gave informed consent. Fetuin-B was measured employinga commercial sandwich enzyme-linked immunosorbent assay. Serum fetuin-B wasdetermined as duplicates in 5 male (34 ± 14.6 years) and 11 female volunteers(29.4 ± 4.1 years) as well as in female volunteers on hormonal contraception(30.0 ± 6.5 years). The duplicate standard deviation was 4.0 ± 2.3%. Thecontraceptive drugs were mono or combined preparations containing 0-0.03 mgethinyl estradiol, and 0.15-3.0 mg of various progestins. In addition, serumfetuin-B was determined as triplicates in 27 female patients undergoingconventional IVF (19) or ICSI (8). The triplicate standard deviation was 3.3 ±1.8%. IVF was declared as 'successful', if at least one oocyte was fertilized,and 'unsuccessful', if no oocyte could be fertilized. Patient age was 34.4 ±4.4 years in successful IVF, and 35.4 ± 3.3 years in unsuccessful IVF. Serumand follicular fluid of patients undergoing controlled ovarian hyperstimulationwere analyzed. Serum was drawn at the day of follicle aspiration.

MAINRESULTS AND THE ROLE OF CHANCE  Serum fetuin-B and follicular fluid fetuin-B were notsignificantly different in six out of nine patients suggesting, in principle,free exchange of fetuin-B between serum and follicular fluid. Thus serumfetuin-B may be used as a proxy of follicular fluid fetuin-B. Serum fetuin-Bincreased during successful IVF cycles (n = 15, P < 0.0001), but did notchange in unsuccessful IVF cycles (n = 6, P = 0.118) despite increasedestradiol levels (P = 0.0019 and P = 0.0254, respectively).

LIMITATIONS,REASONS FOR CAUTION  Thefemale volunteers self-reported their respective hormone medication. Medicationwas verified by serum estradiol, LH and progesterone measurements. For oocyteharvesting, the vaginal wall was punctured once only to minimize co-morbidity.Low grade cross-contamination of individual follicular fluid aspirates andcontamination of the follicular fluid with small amounts of blood wereinevitable. Samples were routinely checked for the presence of hemoglobin thatwould suggest blood contamination. Only samples containing <250 erythrocyteequivalents/µl were used for analysis.

WIDERIMPLICATIONS OF THE FINDING  Serumfetuin-B may be used as a marker to predict the fertilization success in IVF.Fetuin-B levels attained during IVF stimulation may help to make an informeddecision whether oocytes should be fertilized by IVF or by ICSI to overcome thezona pellucida as a barrier.

KEY WORDS：fetuin-B / infertility / IVF fertilizationrate / zona pellucida hardening / follicular fluid / early embryo development/sperm attachment/ embryo hatching

研究假设  在体外受精（IVF）治疗中，血清胎球蛋白B（fetuin-B）含量会与受精率相关吗？

回答总结  IVF过程中，当卵母细胞受精时其血清fetuin-B水平上升，但受精失败时fetuin-B水平不变。

已知  缺乏fetuin-B会导致小鼠透明带蛋白酶ovastacin介导的透明带过早硬化。因此fetuin-B缺乏可造成雌性不孕。

研究设计，样本量，时长  研究人员确定血清fetuin-B正常参考范围，共持续一个月的研究，参与者是5名男性，7名未接受过激素治疗的女性和其他4名服用过不同口服避孕药的女性。收集血样样本后检测血清fetuin-B，黄体生成素（LH），雌二醇和孕酮的含量。另外，由8名进行卵胞浆内单精子显微注射技术（ICSI）的和19名使用重组人卵泡刺激素（rFSH）和/或经历联合使用FSH与LH刺激排卵后接受IVF（21个IVF周期）的女性来测定fetuin-B的正常参考值。每个IVF周期至少取样3次。为比较血清fetuin-B和卵泡液fetuin-B的含量，我们对总体卵泡液中和个体卵泡中的fetuin-B进行分别检测。取样从2012年1月开始，至2014年3月结束。

参与者和研究方法  所有参与者和患者已知情同意。使用酶联免疫吸附试验测定fetuin-B。对5名男性(34 ±14.6岁)、11名女性参与者(29.4 ± 4.1岁)以及9名口服避孕药的女性（30.0 ± 6.5岁）进行重复检测血清fetuin-B含量。重复的标准偏差为4.0 ± 2.3%。口服避孕药含有以下一种或多种成分：0-0.03mg乙烯雌二醇和0.15-3.0 mg各种类型的黄体酮。另外，对27名IVF或ICSI女性中重复测定三次血清fetuin-B。三次重复标准偏差为3.3 ± 1.8%。成功IVF是指至少一个卵母细胞受精，而未成功IVF则指无卵母细胞受精。成功IVF者的平均年龄为34.4 ± 4.4岁，未成功者年龄为35.4 ± 3.3岁。对监控下排卵刺激的患者的血清与卵泡液进行深一步分析。卵泡和血清提取在同一天。

主要结果和可能性  在6/9名患者中其血清和卵泡液的fetuin-B水平无显著性差异，表明血清与卵泡液中fetuin-B可自由进出。因此血清fetuin-B水平可替换卵泡液fetuin-B水平。成功IVF者中fetuin-B水平显著升高（n = 15, P < 0.0001），但在未成功IVF者中fetuin-B无变化（n = 6, P = 0.118），即便雌二醇水平不同（P 分别是0.0019和 0.0254）。

局限性和注意点  女性参与者对自己的激素药物进行自愿报告。通过血清雌二醇，LH和孕酮测定来确认服药。取卵时为减小已存在情况的影响，仅对阴道壁穿刺一次。但是轻微的个体间卵泡液交叉感染或卵泡液与血清的交叉感染是不可避免的。对卵泡液样本进行定期的血红蛋白含量检测来排除血液感染。分析中仅包含红细胞小于250个/ul的参与者。

结果和意义  血清fetuin-B含量可作为预测IVF成功与否的标记物。在IVF过程中对fetuin-B含量进行测定，有助于对IVF或ICSI过程中卵母细胞是否因克服了透明带障碍而成功受精进行了解。

关键词  胎球蛋白B/不孕/体外受精率/透明带硬化/卵泡液/早期胚胎发育/精子附着/胚胎孵出