瘦素对人精子冻融后的保护作用

doi:10.3877/cma.j.issn.1674-0785.2020.02.001

目的

通过在人精子冷冻保护液中添加瘦素，探讨瘦素对冻融后精子质量和功能的作用。

方法

收集2017年1月至12月内蒙古医科大学第三附属医院生殖医学中心就诊患者的精液。前向运动精子比率≥32%且精子浓度≥15×10⁶/ml为正常组，前向运动精子比率<32%且精子浓度<15×10⁶/ml为少弱精组，2组标本各30例。液化后的精液样本分别与含有瘦素的冷冻保护液和不含有瘦素的冷冻保护液混匀。冷冻解冻复苏前后，进行精子活力、存活率以及DNA碎片指数（DFI）的检测，除此以外，检测精子的丙二醛含量及活性氧水平，采用单因素方差分析分析精子的活力、存活率、DFI、丙二醛含量和活性氧水平的差异，组间两两比较采用LSD-t检验。

结果

当添加浓度为10 ng/ml的瘦素后，精子冷冻复苏后总活力最高，达到（53.7±1.5）%。正常组精子在冻融后与新鲜精子相比总活力［（41.5±6.9）% vs （69.9±6.3）%］、存活率［（51.8±4.3）% vs （74.7±3.7）%］均显著下降、DFI［（12.9±3.4）% vs （10.3±3.8）%］上升，活性氧水平［（18.3±1.9）荧光强度vs（7.6±2.6）荧光强度］和丙二醛含量［（5.5±0.8）nmol/10⁸个精子vs（2.6±0.3）nmol/10⁸个精子］均显著上升，差异均具有统计学意义（t=15.7、22.0、2.9、18.2、19.1，P<0.001、<0.001、=0.006、<0.001、<0.001）。少弱精组精子在冻融后与新鲜精子相比总活力［（17.9±2.9）% vs （33.4±5.5）%］、存活率［（39.5±4.4）% vs （57.4±6.2）%］均显著下降、DFI［（35.2±3.6）% vs （21.3±4.1）%］显著上升，活性氧［（64.9±2.9）荧光强度vs（28.3±3.5）荧光强度］和丙二醛［（33.6±2.0）nmol/10⁸个精子 vs （12.1±2.3）nmol/10⁸个精子］均显著上升，差异均具有统计学意义（t=13.6、12.9、13.9、44.4、38.7，P均<0.001）。当添加瘦素后，正常组精子在冷冻解冻后，与对照组相比总活力［（54.7±5.9）% vs （41.5±6.9）%］、存活率［（66.3±3.4）% vs （51.8±4.3）%］均显著上升，活性氧［（10.6±2.1）荧光强度vs（18.3±1.9）荧光强度］和丙二醛［（3.4±0.5）nmol/10⁸个精子 vs （5.5±0.8）nmol/10⁸个精子］显著下降，差异均具有统计学意义（t=7.9、14.4、15.1、12.8，P均<0.001），但DFI比较，组间差异无统计学意义（P>0.05）。少弱精组精子在冷冻解冻后，与对照组相比总活力［（24.8±2.0）% vs （17.9±2.9）%］、存活率［（49.5±3.8）% vs （39.5±4.4）%］均显著上升，DFI［（26.7±2.7）% vs （35.2±3.6）%］、活性氧［（37.9±4.2）荧光强度 vs （64.9±2.9）荧光强度］和丙二醛［（23.1±1.7）nmol/10⁸个精子 vs （33.6±2.0）nmol/10⁸个精子］均显著下降，差异均具有统计学意义（t=10.5、9.4、10.2、28.5、21.9，P均<0.001）。

结论

添加瘦素能有效得减少精子因低温贮藏导致的活力与存活率的下降、DNA碎片及氧化损伤，从而改善精子质量和功能。

关键词: 瘦素, 精子冻融, DNA碎片, 氧化应激损伤, 精子功能

Objective

To investigate the effect of leptin on the quality and function of sperm after freezing and thawing by adding leptin to human sperm cryoprotective solution.

Methods

Semen samples were collected at the Reproductive Medicine Center of the Third Affiliated Hospital of Inner Mongolia Medical University from January to December 2017. Samples with a progressive sperm ratio≥32% and sperm concentration ≥15×10⁶/ml were included in a normal group, while those with a progressive sperm ratio<32% and sperm concentration <15×10⁶/ml were included in an asthenozoospermia (AS) group. The liquefied semen samples were mixed with cryopreserved solution with and without leptin. Vitality rate, motility rate, and DNA fragmentation index (DFI) were measured. Malondialdehyde content and reactive oxygen level in sperm were detected. Comparisons among groups were analyzed by single factor analysis of variance, and comparisons between groups were performed by the LSD-t test.

Results

When leptin was added at a concentration of 10 ng/ml, the total vitality of sperm after cryo-resuscitation was the highest, reaching (53.7±1.5)%. Compared with fresh sperm, the total vitality rate [(41.5±6.9)% vs (69.9±6.3)%, t=15.7, P<0.001] and motility rate [(51.8±4.3)% vs (74.7±3.7)%, t=22.0, P<0.001] of sperm after freezing and thawing in the normal group decreased significantly, while DFI [(12.9±3.4)% vs (10.3±3.8)%, t=2.9, P=0.006], reactive oxygen species (fluorescence intensity, 18.3±1.9 vs 7.6±2.6, t=18.2, P<0.001), and malondialdehyde (nmol/10⁸ sperm, 5.5±0.8 vs 2.6±0.3, t=19.1, P<0.001) all increased significantly. Compared with fresh sperm, the total vitality rate [(17.9±2.9)% vs (33.4±5.5)%, t=13.6, P<0.001] and motility rate [(39.5±4.4)% vs (57.4±6.2)%, t=12.9, P<0.001] decreased significantly in the AS group, while DFI [(35.2±3.6)% vs (21.3±4.1)%, t=13.9, P<0.001], reactive oxygen species (fluorescence intensity, 64.9±2.9 vs 28.3±3.5, t=44.4, P<0.001), and malondialdehyde (nmol/10⁸ sperm, 33.6±2.0 vs 12.1±2.3, t=38.7, P<0.001) all increased significantly. When leptin was added, compared with the control group, the total vitality rate [(54.7±5.9)% vs (41.5±6.9)%, t=7.9, P<0.001] and motility rate[(66.3±3.4)% vs (51.8±4.3)%, t=14.4, P<0.001] of sperm after freezing and thawing in the normal group increased significantly, and reactive oxygen species (fluorescence intensity, 10.6±2.1 vs 18.3±1.9, t=15.1, P<0.001) and malondialdehyde (nmol/10⁸ sperm, 3.4±0.5 vs 5.5±0.8, t=12.8, P<0.001) decreased significantly, although there was no significant difference in DFI between the two groups (P>0.05). After freezing and thawing, compared with the control group, the sperm in the AS group had significantly increased total viability [(24.8±2.0)% vs (17.9±2.9)%, t=10.5, P<0.001] and motility rate[(49.5±3.8)% vs (39.5±4.4)%, t=9.4, P<0.001], and significantly decreased DFI [(49.5±3.8)% vs (39.5±4.4)%, t=10.2, P<0.001], active oxygen (fluorescence intensity, 37.9±4.2 vs 64.9±2.9, t=28.5, P<0.001), and malondialdehyde (nmol/10⁸ sperm, 23.1±1.7 vs 33.6±2.0, t=21.9, P<0.001).

Conclusion

The addition of leptin can effectively reduce sperm motility and viability, DNA fragmentation, and oxidative damage caused by cryopreservation, thereby improving sperm quality and function.

Key words: Leptin, Freezing-thawing, DNA fragmentation, Oxidative stress damage, Sperm function

表1 精液冷冻复苏前后精子的活力、存活率、DFI的比较（±s）

组别		例数	总活力（%）	存活率（%）	DFI（%）
正常组		?	?	?	?
?	冷冻前鲜精	30	69.9±6.3	74.7±3.7	10.3±3.8
?	对照组	15	41.5±6.9^a	51.8±4.3^a	12.9±3.4^a
?	试验组	15	54.7±5.9^b	66.3±3.4^b	11.7±2.2^b
F值		?	136.737	53.325	5.161
P值		?	<0.001	<0.001	0.008
少弱精组		?	?	?	?
?	冷冻前鲜精	30	33.4±5.5	57.4±6.2	21.3±4.1
?	对照组	15	17.9±2.9^c	39.5±4.4^c	35.2±3.6^c
?	试验组	15	24.8±2.0^d	49.5±3.8^d	26.7±2.7^d
F值		?	125.810	99.784	118.606
P值		?	<0.001	<0.001	0.008

表1 精液冷冻复苏前后精子的活力、存活率、DFI的比较（±s）

组别		例数	总活力（%）	存活率（%）	DFI（%）
正常组		?	?	?	?
?	冷冻前鲜精	30	69.9±6.3	74.7±3.7	10.3±3.8
?	对照组	15	41.5±6.9^a	51.8±4.3^a	12.9±3.4^a
?	试验组	15	54.7±5.9^b	66.3±3.4^b	11.7±2.2^b
F值		?	136.737	53.325	5.161
P值		?	<0.001	<0.001	0.008
少弱精组		?	?	?	?
?	冷冻前鲜精	30	33.4±5.5	57.4±6.2	21.3±4.1
?	对照组	15	17.9±2.9^c	39.5±4.4^c	35.2±3.6^c
?	试验组	15	24.8±2.0^d	49.5±3.8^d	26.7±2.7^d
F值		?	125.810	99.784	118.606
P值		?	<0.001	<0.001	0.008

表2 精液冷冻复苏前后精子活性氧和丙二醛水平的比较（±s）

组别		例数	活性氧（荧光强度）	丙二醛（nmol/10⁸）
正常组		?	?
?	冷冻前鲜精	30	7.6±2.6	2.6±0.3
?	对照组	15	18.3±1.9^a	5.53±0.8^a
?	试验组	15	10.6±2.1^b	3.4±0.5^b
F值		?	185.411	222.766
P值		?	<0.001	<0.001
少弱精组		?	?
?	冷冻前鲜精	30	28.3±3.5	12.1±2.3
?	对照组	15	64.9±2.9^c	33.6±2.0^c
?	试验组	15	37.9±4.2^d	23.1±1.7^d
F值		?	823.525	859.001
P值		?	<0.001	<0.001

表2 精液冷冻复苏前后精子活性氧和丙二醛水平的比较（±s）

组别		例数	活性氧（荧光强度）	丙二醛（nmol/10⁸）
正常组		?	?
?	冷冻前鲜精	30	7.6±2.6	2.6±0.3
?	对照组	15	18.3±1.9^a	5.53±0.8^a
?	试验组	15	10.6±2.1^b	3.4±0.5^b
F值		?	185.411	222.766
P值		?	<0.001	<0.001
少弱精组		?	?
?	冷冻前鲜精	30	28.3±3.5	12.1±2.3
?	对照组	15	64.9±2.9^c	33.6±2.0^c
?	试验组	15	37.9±4.2^d	23.1±1.7^d
F值		?	823.525	859.001
P值		?	<0.001	<0.001

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Protective effect of leptin on human sperm after cryopreservation

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Protective effect of leptin on human sperm after cryopreservation