Preliminary Study of Human Follicular Fluid Fatty Acid Spectrum on Assessing the Developmental Potential of Oocytes

目的:分析体外受精(IVF)患者单卵泡液脂肪酸谱表达差异与卵母细胞发育潜能的相关性.方法:利用气相色谱分析技术检测卵泡液脂肪酸谱,以第3日胚胎碎片评分(EFS)及胚胎细胞数目(ECN)为卵母细胞发育结局的评价指标,行相关性分析.结果:单卵泡液中普遍存在6种脂肪酸:十一烷酸、肉蔻豆酸、棕榈酸、硬脂酸、油酸和亚油酸.Spearman分析显示油酸与EFS呈正相关(r=0.357,P=0.014),各种脂肪酸与ECN无明显相关性.Logistic回归进一步分析显示油酸浓度为EFS阳性预测因子(OR=1.035,95％CI 1.007～1.064).结论:人卵泡液油酸含量在一定程度上可预测卵母细胞发育潜能.     

卵母细胞分泌的生长分化因子-9和骨形成蛋白-15与卵母细胞发育潜能的关系

目的:探讨卵母细胞分泌的生长分化因子-9(GDF-9)和骨形成蛋白-15(BMP-15)与卵母细胞发育潜能的关系。方法:收集因输卵管性不孕接受IVF-ET治疗患者的卵泡,每侧卵巢穿刺取卵时首先选择一个直径≥18 mm卵泡抽吸,对卵母细胞进行成熟度分型,并观察其受精、卵裂及胚胎评分情况。受精后按是否形成优质胚胎将将卵泡分为优质卵母细胞组和非优质卵母细胞组,采用Western blotting方法检测比较各组卵泡液中GDF-9和BMP-15的含量。结果:①优质卵母细胞组的GDF-9蛋白相对含量为2.498±0.133,非优质卵母细胞组为1.305±0.406,差异有统计学意义(P=0.008)。②优质卵母细胞组的BMP-15蛋白相对含量为3.379±0.482,非优质卵母细胞组为1.336±0.214,差异有统计学意义(P=0.003)。结论:在优质卵母细胞组卵泡液中GDF-9和BMP-15水平均较高,提示人卵泡液中GDF-9、BMP-15水平可以反映卵母细胞的发育潜能,GDF-9、BMP-15水平可以作为评价卵母细胞质量的指标。     

子宫内膜异位症相关性不孕卵泡液代谢物组初筛及其与卵母细胞质量的关系

目的探讨与子宫内膜异位症(EMS)相关性不孕患者卵母细胞发育潜能相关的代谢物组。方法利用超高效液相色谱-质谱(HPLC-MS)对接受体外受精(IVF)的EMS相关性不孕患者即EMS组(n=27)和同期因男方因素行体外受精的健康妇女即对照组(n=28)进行卵泡液代谢组学分析。结果共观察到与EMS相关性不孕密切相关的差异代谢物28种,主要有脂类、核苷酸类、蛋白降解片段及其它等四大类,其中上调的代谢物主要有Valyl-Arginine、Methyl-15-cyanopentadecanoate、4-Oxo-13-cis-retinoate等15种;下调的代谢物主要有Menadiol dibutyrate、3-hydroxynonanoyl carnitine、Phytosphingosine等13种。结论本研究发现并描绘出两组卵泡液差异性代谢物谱,这28种代谢物组可能成为预测EMS妇女卵母细胞早期发育潜能的重要生物标志物。然而,并未见其与随后的胚胎质量、累积妊娠率以及活产率间明显的相关性。     

卵泡液和卵裂期胚胎培养液可溶性人类白细胞抗原G(sHLA-G)的表达与IVF-ET结局的相关性研究

目的:探讨卵泡液和卵裂起胚胎培养液中可溶性人类白细胞抗原G(sHLA-G)与卵裂期胚胎发育以及IVF-ET后临床妊娠率的关系,为临床优选卵母细胞及移植胚胎提供一种有效的非侵入性检测途径。方法:采用酶联免疫吸附试验(ELISA)检测取卵日卵泡液及第3日移植胚胎培养液中sHLA-G的表达及表达量,将40例患者分为sHLA-G表达阳性组(n=15)和sHLA-G表达阴性组(n=25)进行比较分析。结果:40份患者卵泡液sHLA-G阳性率为37.5%(15/40),87份第3日移植胚胎培养液中sHLA-G阳性率为56.32%(49/87),卵泡液和胚胎培养液sHLA-G阳性组与阴性组的获卵数和受精率均无差异(P>0.05),sHLA-G阳性组的卵裂率、第3日胚胎卵裂球数、优质胚胎率、临床妊娠率均明显高于阴性组(P<0.05),其中胚胎培养液sHLA-G阳性组的临床妊娠率显著高于阴性组(P<0.01)。结论:sHLA-G在部分卵泡液及早期胚胎培养液中均有表达,反映了胚胎良好的发育和种植潜能,预示着较好的IVF-ET结局,是一种优选卵母细胞和移植胚胎的有效的非侵入性途径。     

卵泡液代谢组学最新研究进展

代谢组学是继基因组学、转录组学、蛋白质组学之后系统生物学的主要研究平台,并逐渐成为生命科学的研究热点。在辅助生殖技术(assisted reproductive technology,ART)领域,卵泡液代谢组学已取得较大进展。目前,国内外大多数回顾性研究表明卵泡液相关代谢物(糖类、脂肪酸、氨基酸、激素等)可预测卵母细胞发育潜能,但有待大样本、多中心随机对照试验进一步证实。卵泡液代谢组学的深入研究有望成为评估卵母细胞质量并改善ART治疗结局的重要方法和手段。     

妊娠与非妊娠病人的卵泡液对体外培养鼠胚发育的影响

探究卵泡液的成份与卵母细胞成熟及活力之间的关系,结果令人失望;卵泡液中的类固醇激素、蛋白激素与卵母细胞成熟抑制物等成份,因变化太大而在体外受精(IVF)中不能有预测的价值。本研究是应用培养基中再加IVF时所抽取的人卵泡液对单细胞鼠胚作体外培养,观察鼠胚发育结果,以确定在人卵泡液中可能存在的有关影响卵母细胞受精与胚胎存活力的物质。在实验一中,组一用HTF培养基加入来自产生受精胚胎的卵泡液按2%、5%、10%与15%(V/V)浓度培养单细胞实验鼠胚;在组二中用     

卵泡液中雌孕激素水平及卵泡大小与卵母细胞成熟度的关系

目的:探讨卵胞浆内单精子显微注射(ICSI)周期取卵日卵泡大小、卵泡液中雌孕激素水平和卵母细胞成熟度的相关性。方法:选择因男方因素在本中心接受ICSI治疗的51例不孕患者作为研究对象,收集取卵日卵泡液共140例,取卵前B超测量卵泡平均直径,采用电化学发光法(ECLI)检测卵泡液中雌二醇(E2)、孕酮(P)水平,分析取卵日卵泡大小、卵母细胞成熟度及卵泡液中雌孕激素水平三者间的相互关系。结果:卵泡平均直径与卵泡液E2水平呈正相关(r=0.724,P<0.05),与P水平亦呈正相关(r=0.798,P<0.05);按卵母细胞成熟度分为MⅡ组、MⅠ组、GV组和退化组,4组卵泡液中P水平无统计学差异(P>0.05),但退化组卵泡液中E2水平显著低于MⅡ组(P<0.05);所有样本按P/E2值分为1(<15)、2(15～25)、3(>25)组,2组卵母细胞成熟率最高,与1、3组的差异均有统计学意义(P<0.017),1组与3组的卵母细胞成熟率无统计学差异(P>0.017)。根据卵泡平均直径分组,直径>1.6cm为大卵泡组,1.6～1.2cm为中卵泡组,<1.2cm为小卵泡组,大卵泡组卵母细胞成熟率最高,与中、小卵泡组的差异有统计学意义(P<0.017),中卵泡组卵母细胞成熟率显著高于小卵泡组(P<0.017)。结论:卵泡大小与卵泡液雌孕激素水平呈正相关。卵泡液中P/E2在特定范围内时卵母细胞成熟率较高,其水平过高或过低均影响卵母细胞成熟;大卵泡组与中、小卵泡组在卵母细胞成熟率方面有显著统计学差异。     

边缘性空卵泡综合征1例

正空卵泡综合征(empty follicle syndrome,EFS)是指经控制性超促排卵(controlled ovarian hyperstimulation,COH)后,卵巢有多个正常卵泡发育,血清雌二醇(E_2)水平正常,但取卵手术时经反复冲洗及抽吸仍不能从成熟卵泡中获得卵母细胞。而边缘性EFS(borderline form of EFS)是指在COH后,诱发排卵前尽管有较多卵泡发育,但取卵时仅获得少量成熟卵母细胞或不成熟卵母细胞。此两种病例临床皆少见,一旦出现则不得不取消周期,给患者带来不小的经济和心理负担。我院发现疑似边缘性EFS 1例,现报道如下。     

反复多周期促排卵对小鼠母体卵巢组织及胚胎发育潜能的影响

目的:比较多周期连续促排卵对小鼠卵巢组织和胚胎发育潜能的影响。方法:建立反复多周期促排卵小鼠模型(A组),观察其卵巢组织内各级卵泡数形态并计数。统计获取的卵母细胞及受精卵数、卵裂率、优质胚胎率和囊胚形成率,并与单次促排卵周期小鼠(B组)、自然周期排卵小鼠(C组)比较。结果:B组获取的卵母细胞数及受精卵数显著增多(41.6±11.0)。而A组获取的卵母细胞及受精卵数显著降低(5.5±2.9),但初级卵泡形态异常率(33.34%)和次级卵泡形态异常率(27.14%)显著高于C组(8.33%、5.62%)与B组(10.34%、8.97%)(P<0.01);卵裂率(44.83%)、优质胚胎率(0)和囊胚形成率(0)均显著低于C组(88.07%、75.09%、74.74%)和B组(81.05%、69.02%、66.94%)(P<0.01)。结论:单次促排卵能增加卵母细胞和胚胎的数量。但多次连续促排卵会增加卵巢内异常的初级卵泡和次级卵泡数,降低卵母细胞质量。同时,会降低优质胚胎率和囊胚形成率,影响卵裂后胚胎进一步发育的潜能。     

PCOS患者卵泡液内IGF-Ⅰ的水平对卵母细胞成熟的影响

目的:探讨PCOS患者卵泡液内胰岛素样生长因子-Ⅰ(IGF-Ⅰ)的水平与卵母细胞发育成熟的关系。方法:选取因PCOS接受IVF治疗的患者41例为PCOS组、同期因输卵管因素接受治疗的患者37例为对照组。在取卵日根据卵泡直径进行分组,分为≤14mm组和>14mm组,每例患者留取不同直径3～4个卵泡的卵泡液。分别检测PCOS组和对照组患者卵泡液内IGF-Ⅰ的水平,并分析其与卵母细胞成熟和受精的关系。结果:PCOS组IGF-Ⅰ、雌二醇(E2)、睾酮(T)的水平显著高于对照组(P<0.05),与卵泡直径不相关。在卵泡直径≤14mm组,PCOS患者卵母细胞成熟率和受精率明显高于对照组(P<0.05)。在卵泡直径>14mm组,PCOS组与对照组间卵母细胞成熟率和受精率无差异性。PCOS组卵泡液内IGF-Ⅰ水平和E2、T呈正相关(P<0.05)。结论:PCOS患者促排卵过程中卵泡液内IGF-Ⅰ水平的升高与卵泡直径无相关性,而卵泡液内高IGF-Ⅰ水平可影响PCOS患者小于14mm卵泡的卵母细胞成熟和受精。     

Fatty acids of follicular fluid phospholipids and triglycerides display distinct association with IVF outcomes

Research questionAre triglyceride fatty acids in the follicular fluid associated with either follicular fluid phospholipid fatty acids or IVF outcomes and, if so, how are they associated?DesignIn a prospective cross-sectional study, 70 women undergoing intracytoplasmic sperm injection were recruited. Follicular fluid phospholipids and triglycerides were separated by thin-layer chromatography. Fatty acids were measured using gas-liquid chromatography and flame ionization detection system.ResultsSignificant differences in fatty acid composition were observed between follicular fluid phospholipid and triglyceride fractions. Phospholipid stearic acid and n-3 polyunsaturated fatty acids, particularly alpha-linolenic acid, were negatively associated with the number of mature oocytes and cleaved embryos, whereas arachidonic acid was in direct correlation with cleavage rate per IVF cycle (β = 0.325, P = 0.022). In the case of triglyceride fraction, total monounsaturated fatty acids, oleic acid in particular, displayed significantly positive associations with the number of oocytes (β = 0.261, P = 0.043) and embryos (β = 0.310, P = 0.018). Furthermore, cleavage rate correlated inversely with palmitic acid (β = –0.359, P = 0.007) and directly with pentadecanoic acid (β = 0.378, P = 0.005). Most of these associations, however, were not independent of predictive fatty acids belonging to phospholipid fraction, according to multivariate analysis.ConclusionsFatty acid compositions of phospholipid and triglyceride fractions from human follicular fluid differentially correlate with IVF cycle parameters.     

Amino acid and fatty acid composition of follicular fluid as predictors of in-vitro embryo development

The value of using the amino acid and fatty acid composition of follicular fluid as predictors of embryo development was assessed in a bovine model of in-vitro maturation (IVM), IVF and blastocyst culture (IVC). A total of 445 cumulus-oocyte complexes (COC) aspirated from visually healthy follicles underwent IVM and IVF singly (n = 138) or in groups (n = 307). Of these COC, 349 cleaved (78%) following IVF and 112 went on to form blastocysts (32% of cleaved) following IVC. Culture method (singly or in groups) had no effect on development. In contrast to fatty acids, which had no predictive value, the amino acid composition of follicular fluid was associated with morphological assessments of COC quality and with post-fertilization development to the blastocyst stage. Principal component analysis identified two amino acids (i.e. alanine and glycine) that had the highest value for predicting early post-fertilization development. The predictive value of these two amino acids, in terms of the percentage of oocytes that cleaved following IVF, was greatest for COC with the poorest morphological grades but, with respect to blastocyst yields, was independent of morphological grade, and so may serve as a useful additional non-invasive measure of COC quality.     

The association between microenvironmental reactive oxygen species and embryo development in assisted reproduction technology cycles

This study was designed to determine the relevance between the levels of reactive oxygen species (ROS) in microenvironment (follicular fluid or culture media) and the embryo development in IVF/ICSI cycles. A total of 466 follicles from 174 IVF/ICSI cycles were collected for this study. The ROS levels in monofollicular fluid and spent culture media were evaluated by chemiluminescence assay with luminol as a probe. The results demonstrated that it is in ICSI cycles that elevated ROS levels in follicular fluid were associated with day 3 poor embryo quality. The ROS levels in spent culture media were correlated with advanced degree of fragmentation. In addition, ROS levels in culture media, instead of in follicular fluid, were negatively correlated with implantation potential of embryos. The ROS levels in culture media may be viewed as an embryo metabolic marker and function as an adjuvant criterion for embryo selection.     

溶血磷脂酸在卵母细胞体外成熟过程中作用的研究进展

卵母细胞体外成熟(IVM)已成为一项热门的辅助生殖技术(ART),有广泛的应用和发展前景。但其体外成熟率和受精后的发育能力还有待提高,培养条件需要进一步优化。溶血磷脂酸(LPA)是磷脂激素家族的重要成员,有生长因子和激素样活性,可产生广泛的生物学效应。研究表明LPA存在于卵泡液中,对卵泡发育、卵母细胞IVM、受精、胚胎发育、种植等产生重要作用。本文就LPA特性及其在卵母细胞体外成熟过程中的作用做一简要的阐述。     

Changes in fatty acid composition of cervical mucus lecithin during pregnancy

The fatty acid composition in cervical mucus was determined in 37 pregnant and 17 nonpregnant women using gas liquid chromatography. In both groups palmitic acid (16:0) and oleic acid (18:1) were the predominant acids comprising more than half of the total amount. Compared to nonpregnant controls, pregnant women had markedly elevated levels of oleic acid (18:1), while mean levels of myristic acid (14:0) and stearic acid (18:0) were significantly lower. These pregnancy-induced changes in fatty acid pattern could not be ascribed to the increased viscosity of cervical mucus. Only minor differences in fatty acid pattern were found between women in first trimester and at term. In contrast to nonpregnant controls, a significant negative correlation between levels of myristic acid (14:0) and docosahexaenoic acid (22:6) was found in pregnant women, indicating a redistribution of these two fatty acids during pregnancy. The present study demonstrates that pregnancy alters the fatty acid composition of cervical mucus lecithin, and that the characteristic changes are present before the 10th gestational week.     

Perifollicular blood flow Doppler indices, but not follicular pO2, pCO2, or pH, predict oocyte developmental competence in in vitro fertilization.

OBJECTIVE: To assess the relationships among perifollicular blood flow; follicular fluid pO2, pCO2, and pH; oocyte developmental capacity; preimplantation embryo quality. DESIGN: Prospective study. SETTING: Academic, tertiary care institution. PATIENT(S): Unselected, gonadotropin-stimulated IVF cycles. INTERVENTION(S): Color, pulsed Doppler analysis of perifollicular blood flow, and follicular pO2, pCO2, and pH determinations of randomly designated, mapped ovarian follicles. MAIN OUTCOME MEASURE(S): Fertilization and day 3 embryo cleavage and morphology. RESULT(S): Perifollicular vascularity indices were significantly and negatively correlated with day 3 embryo cleavage. Pulsatility index and S-D ratio also were significantly and negatively correlated with follicular pO2. The same correlation was found between resistance index and the fertilization rate of preovulatory oocytes. No relationship existed between follicular metabolic analysis and fertilization or embryo quality. The resistance index had a sensitivity of 0.57 and a specificity of 0.71 for the prediction of advanced embryo cleavage status. CONCLUSION(S): Results confirm and extend previous reports demonstrating that color, pulsed Doppler ultrasound analysis of individual preovulatory follicles during IVF therapy may provide an indirect index of the developmental competence of the corresponding oocyte. Although these methods may provide means to select embryos for transfer with the highest implantation potential, the moderate predictive power showed so far may limit their clinical applicability.     

Vascular endothelial growth factor, nitric oxide, and leptin follicular fluid levels correlate negatively with embryo quality in IVF patients

Objective(s): To measure vascular endothelial growth factor (VEGF), nitric oxide (NO) and leptin levels in individual ovarian follicles and to examine their relationships with perifollicular blood flow, follicular metabolic indices, and the developmental potential of the corresponding oocyte and embryo.

Design: Prospective study.

Setting: Academic, tertiary care institution.

Patient(s): Unselected IVF patients.

Intervention(s): Color-pulsed Doppler analysis of perifollicular blood flow; determination of partial pressure of oxygen (pO₂), partial pressure of carbon dioxide (pCO₂), and pH and VEGF, leptin and NO levels in follicular fluid.

Main Outcome Measure(s): Fertilization and day 3 embryo morphology and cleavage.

Result(s): Fifty-five follicular fluid samples from 16 patients were studied. Mean follicular fluid levels were as follows: VEGF, 1,046 ± 863.7 pg/mL (range, <63–3,332.7 pg/mL); NO₃/NO₂, 34.2 ± 12 μM (range, 16.4–76.1 μM); and leptin, 20.1 ± 12.1 ng/mL (range, 3.3–52.2 ng/mL). Vascular endothelial growth factor had a negative correlation with embryo morphology (r = −0.28, P=.01). Leptin demonstrated a negative correlation with follicular pO₂ (r = −0.42, P=.005) and a positive correlation with follicular pCO₂ (r = 0.36, P=.02). Follicular leptin levels correlated positively with VEGF levels (r = 0.46, P=.008) and with NO₃/NO₂ levels (r = 0.39, P=.006).

Conclusion(s): Vascular endothelial growth factor, NO and leptin appear to be markers of follicular hypoxia and suboptimal embryo development. Whether fluctuations of these regulatory factors determine or reflect changes in the follicular microenvironment affecting oocyte developmental potential remains to be elucidated.     

Dimethyl acetals, an indirect marker of the endogenous antioxidant plasmalogen level, are reduced in blood lipids of Sudanese pre-eclamptic subjects whose background diet is high in carbohydrate

In Sudanese women with (n = 60) and without (n = 65) pre-eclampsia, circulating lipids, plasma and red cell saturated and monounsaturated fatty (MUFA) acids and dimethyl acetals (DMAs) were investigated. DMAs are an indirect marker of levels of plasmalogens, endogenous antioxidants, which play a critical role in oxidative protection, and cholesterol homeostasis. The pre-eclamptics had higher C18:1n-9 (p < 0.001) and ΣMUFA (p < 0.01) in plasma free fatty acids, C16:1n-7, C18:1n-9, ΣMUFA; 16:0/16:1n-7 (p < 0.01) in erythrocyte choline phosphoglycerides (ePC) and 16:1n-7, 18:1n-7 and 16:0/16:1n-7 (p < 0.01) in erythrocyte ethanolamine phosphoglycerides (ePE). In contrast, the DMAs 18:0, 18:1 and ΣDMAs in ePE, and 16:0, 18:0 and ΣDMAs in ePC were reduced (p < 0.001) in the pre-eclamptic women. This study of pregnant women with high carbohydrate and low fat background diet suggests pre-eclampsia is associated with oxidative stress and enhanced activity of the microsomal enzyme stearyl-CoA desaturase (delta 9 desaturase), as assessed by palmitic/palmitoleic (C16:0/C16:n-1) and stearic/oleic (C18/C18:1n-9) ratios.     

Fatty acid composition of mid-trimester amniotic fluid in women of different ethnicities

Objective: To determine whether the fatty acid composition of mid-trimester amniotic fluid differs by ethnicity and pregnancy outcome. Methods: Fatty acid composition was analyzed by gas chromatography in 198 women undergoing amniocentesis at 15–19 weeks gestation. Cytokine levels were determined by ELISA in a subgroup of 52 subjects. Results: The major fatty acids detected were palmitic acid (31.8%) and stearic acid (31.5%). The n-6 polyunsaturated fatty acids (PUFA), linoleic acid (LA, 18:2) and arachidonic acid (AA, 20:4), were 11.3%, while the n-3 PUFA fatty acids, α linolenic acid (ALA, 18:3) and docosahexaenoic acid (DHA, 22:6), were 3.8% of the total. Palmitic acid was a higher percentage in Asians (40.5%) and Whites (34.5%) than in Blacks (22.2%) and Hispanics (23.7%) (p?≤?0.0012). Oleic acid (18:1 n-9) was a higher percentage in Blacks (12.2%) and Hispanics (12.1%) than in Whites (9.2%) or Asians (7.5%) (≤0.0002). LA and AA were higher in Blacks (9.0%, 5.4%) and Hispanics (8.6%, 4.1%) than in Whites (6.1%, 3.7%) and Asians (5.5%, 2.9%) (p?≤?0.0002). DHA did not differ among the ethnic groups or according to pregnancy outcome. A reduced palmitic acid percentage was identified in the six women with preeclampsia (p?=?0.0233). Tumor necrosis factor-α levels were inversely proportional to the palmitic acid percentage (p?=?0.0275) and positively associated with the percentages of stearic (18:0) (p?=?0.0132) and oleic (p?=?0.0290) acids. Conclusions: Amniotic fluid fatty acid composition differed among the ethnic groups and may influence inflammatory mediator production and susceptibility to preeclampsia.