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IX 図表
図 1 フローサイトメトリによる代表的なヒストグラム。 Typical case 1 は AIH(人工授精)により妊
娠したカップルにおける男性パートナーの精液の解析結果、Typical case 2 は IVF（体外受精）に
より妊娠したカップルにおける男性パートナーの精液の解析結果。上段(A,B,E,F)は Alexa488 標
識二次抗体蛍光（FL1）に対する側方散乱光（side scatter；SSC）のドットプロットであり、下段
(C,D,G,H)は Alexa488 標識二次抗体蛍光（FL1）のヒストグラムである。また左図（A,C,E,G）は精
子を normal mouse IgG（アイソタイプコントロール）抗体により染色したもの、右図（B,D,F,H）は
抗 SPMI マウス IgG 抗体により染色したものである。SEMG 陽性精子の集団は、抗 SPMI マウス
IgG 抗体染色によるヒストグラム（D,H）と normal mouse IgG 抗体染色によるヒストグラム（C,G）
をそれぞれ比較することによって決定した。
50
図 2 総 SEMG 陰性カウントによる経子宮妊娠予測の AUC-ROC 解析結果
図 3 (a)抗酸化投与または(b)メチルコバラミン投与による精子濃度の改善を予測に対する、治療
前 ORP の AUC－ROC 解析結果
51
表 1 男性不妊群、コントロール群における精液検査所見と SEMG 陽性精子率、SEMG 陰性精
子率
Characteristics
Patients (N = 142)
Mean ± SD
Median Min-max
Control (N = 13)
Mean ± SD Median Min-max
Age (years)
Vol (mL)
Conc (×106/mL)
Mot (%)
36.5 ± 6.6
4.25 ± 1.73
50.6 ± 58.0
27.5 ± 19.9
30.1 ± 4.1
N/A
76.8 ± 7.3
63.4 ± 2.1
35.5
3.75
33.9
25.75
21-60
1.2-9.3
0.0004-400
0-86.1
TSC (×106/mL) 208.7 ± 19.8 143.5
Viability (%)
55.76 ± 19.3 58.7
SEMG+ (%)
71.2 ± 20.2
73.5
0.003-1475 N/A
1.8-87.6
N/A
12.2-99.2
20.0± 11.8
SEMG− (%)
0.8-87.8
28.8 ± 20.2
26.5
29.5
N/A
81.5
61.05
25-36
N/A
45.9-167.8
50.8-86.6
N/A
N/A
18.6
N/A
N/A
6.1-53.2
80.0 ± 11.8 81.4
46.8-93.9
SEMG+: SEMG-positive spermatozoa; SEMG−: SEMG-negative spermatozoa; Patients: male
infertile patients; Control: normal healthy male subjects with pregnant wives.
Mean values of each parameter for patients and control subjects were compared using Wilcoxon rank
sum test, which revealed significant differences concerning all parameters compared.
Vol (semen volume); N/A (not applicable); Conc (sperm concentration); Mot (total sperm motility)
TSC (total sperm count); Viability (sperm viability)
表 2 男性不妊群における SEMG 陽性精子率と各精液パラメーターとの相関
Characteristics
Correlation coefficient (ρ)
P value
Age (years)
Vol (mL)
Conc (×106/mL)
Mot (%)
TSC (×106/mL)
0.00
0.02
-0.12
-0.23
-0.14
0.986
0.838
0.152
0.005
0.098
Viability (%)
FSH (mIU/mL)
LH (mIU /mL)
T (ng/dL)
-0.13
0.13
0.09
0.00
0.134
0.135
0.320
0.985
ρ: Spearman’s rank correlation coefficient.
52
表 3 コントロール群における SEMG 陽性精子率と各精液パラメーターとの相関
Characteristics
Correlation coefficient (ρ)
P value
Age (years)
Sperm concentration (×106/mL)
Sperm motility (%)
0.07
0.104
-0.27
0.835
0.734
0.374
ρ: Spearman’s rank correlation coefficient.
表 4 妊娠群、非妊娠群間でのパラメーターの比較
Characteristics
Pregnancy (N = 36)
Non-pregnancy (N =60)
Mean ± SD
Median Min-max
Mean ± SD
Median Min-max
Age (years)
35 ± 5.8
34.5
21-48
37 ± 6.4
36
26-56
0.24
Vol (mL)
3.9 ± 1.4
3.6
1.2-8
4.0 ± 1.7
3.7
1.2-8.9
0.89
Conc (×106/mL) 49.7 ± 57.0
33.9
0.001-322.8
53.9 ± 68.8
34.8
0.0004-399.9 0.92
Mot (%)
28.8 ± 20.5
26.9
0-86.1
25.6 ± 18.8
24.65
0-66.4
0.43
TSC (×106/mL)
186.3 ± 196.3 145.1
0.005-839.3
279.8 ± 221.0 126.4
0.003-1475
0.89
Viability (%)
57.6 ± 14.8
57.4
21-84.2
55.5 ± 20.1
58.8
1.9-86.6
0.85
FSH (mIU/mL)
4.9 ± 2.7
4.6
1.6-16
5.2 ± 3.8
4.3
1.4-18.8
0.73
LH (mIU /mL)
3.5 ± 1.7
2.8
1.8-7.8
2.9 ± 1.5
2.6
1.3-9.6
0.08
T (ng/dL)
487.7 ± 171.4 480.3
124-1067
463.6± 166.6 448
167-834
0.51
SEMG+ (%)
73.9 ± 18.5
73.2
27.6-99
71.9 ± 19.8
73.8
12.2-96.9
0.70
SEMG- (%)
26.1 ± 18.5
26.8
1-72.4
28.1 ± 19.8
26.2
3.1-87.8
0.70
0-548.0
144.8 ± 185.3 77.3
0-1075
0.83
0-430.2
76.2 ± 143.6 21.0
0.0002-821.5 0.90
Total SEMG+ count
(×106)
127.7 ± 121.6 101.5
Total SEMG− count
P value
(×106)
58.6 ± 96.4
28.7
Note that only 96 of 142 patients answered the questionnaires regarding pregnancy outcomes and were
included in this analysis. Mean values of each parameter for patients and control subjects were compared
using Wilcoxon rank sum test.
53
表 5 経子宮妊娠群、非経子宮妊娠群におけるパラメーターの比較
Characteristics
SP+IUI (N = 20)
IVF+ ICSI (N = 16)
P value
Age (years)
Semen volume (mL)
Sperm concentration (×106/mL)
Sperm motility (%)
Sperm viability (%)
Total sperm count (×106)
Total motile sperm count (×106)
Total viable sperm count (×106)
35 ± 6.1
4.0 ± 1.3
56.2 ± 67.0
31.2 ± 22.3
59.0 ± 12.9
203.6 ± 195.0
88.4 ± 158.2
125.7 ± 136.9
36 ± 5.5
3.8 ± 1.4
41.7 ± 42.0
25.8 ± 18.2
55.7 ± 17.3
163.5 ± 199.4
65.0 ± 120.1
103.6 ± 116.2
0.44
0.74
0.34
0.67
0.91
0.32
0.26
0.48
SEMG+ (%)
69.9 ± 19.4
130.2 ± 103.8
73.4 ± 114.5
78.8 ± 16.6
126.9 ± 149.7
36.7 ± 60.3
0.21
0.34
0.06
Total SEMG+ count
(×106)
Total SEMG− count (×106)
SP+IUI: patients whose wives achieved pregnancy through spontaneously (SP) or IUI; IVF+
ICSI: patients whose wives achieved pregnancy through IVF or ICSI.
54
表 6. 研究 2 における患者背景
Characteristic
Antioxidants
Methylcobalamin
(n = 40)
(n = 37)
Age (y)
36.5 (32-41.8)
Serum hormones
Testosterone† (ng/mL)
4.15 (3.10-5.56)
LH† (IU/L)
4.2 (2.58-5.3)
FSH† (IU/L)
4.46 (3.5-7.55)
Testicular volume† (mL)
Right
16 (14-20)
Left
17 (14-20)
Variables of varicocele
Varicocele laterality (n )
None
29
Unilateral, right
Unilateral, left
Bilateral
Varicocele grade (n)
Right
None
34
Grade 1
Grade 2
Grade 3
Left
None
32
Grade 1
Grade 2
Grade 3
Semen parameters
Semen volume† (mL)
2.75 (1.74-3.4)
Sperm concentration† (n
16.79 (5.81-49.33)
x106/mL)
Sperm motility† (%)
21.35 (9.63-34.10)
Total motile sperm count (n
10.29 (3.24-25.64)
x 106)
ORP† (mV/106 sperm/mL)
1.51 (0.49-5.44)
8-OHdG† (μmol/dL)
10.76 (9.70-12.06)
†Median values (25th–75th percentile),
*Wilcoxon rank-sum test
** χ2 corrected test.
55
36 (32.5-42)
0.610*
4.35 (3.05-5.86)
3.6 (3.1-5.6)
5.42 (4.1-8.3)
0.705*
0.820*
0.506*
18 (14-21.75)
18 (14-21)
0.697*
0.652*
0.825**
28
0.754**
33
0.376**
31
2.45 (1.55-3.3)
15.4 (8.49-31.18)
25.67 (14.67-33.88)
9.11 (2.77-28.82)
1.30 (0.36-2.74)
10.78 (8.46-14.35)
0.361*
0.729*
0.534*
0.737*
0.600*
0.815*
表 7 投与前後での精液検査と OS マーカーの値
Parameters
Baseline
Antioxidants
Methylcobalamin
(n = 36)
3 months
(n = 31†)
3 months
P§
Baseline
P§
P¶
2.75 (1.7-3.4)
2.55 (1.6-3.95) 0.776 2.45 (1.6-3.3)
2 (1.3-4.2)
0.297 0.955
Semen volume‡ (mL)
Sperm concentration‡
16.8(6.0-49.3)
19.1 (8.6-33.8) 0.927 14.0 (5.3-27.6) 19.0 (8.8-31.0) 0.017 0.945
(nx106/mL)
21.4 (8.7-32.9) 20.8(11.2-40.6) 0.036 24.5 (10-32.5)
15.5 (7.2-34.0) 0.261 0.297
Sperm motility‡ (%)
10.3 (3.2-25.9) 9.8 (2.8-23.5)
0.747 8.9 (2.1-14.0)
6.8 (3.1-15.9)
0.462 0.421
TMC‡ (nx106)
ORP‡ (mV/10 sperm/mL) 1.51 (0.40-5.07) 2.04 (0.85-3.33) 0.866 1.95 (0.64-3.70) 3.26 (1.43-6.02) 0.254 0.121
10.7 (8.9-120.6) 10.6 (9.1-13.0) 0.314 10.6 (8.7-14.2) 10.3 (8.9-13.0) 0.80.8560.50.567
8-OHdG‡ (μmol/dL)
†The number of samples used for assessing sperm motility, total motile sperm count, and ORP
were not 31, but only 30, 30, and 29, respectively.
‡Median values (25th–75th percentile).
§Wilcoxon signed-rank sum test.
¶ Wilcoxon rank-sum test.
表 8 低 ORP 群、高 ORP 群間における治療前精液検査と 8-OHdG の比較
Characteristic
Low ORP
High ORP
(n = 52)
(n = 22)
Semen volume‡ (mL)
2.13 (1.51-3.18)
2.95 (2.5-3.86)
Sperm concentration‡ (n x 106/mL) 27.97 (12.31-51.26)
5.71 (4.41-11.65)
Sperm motility‡ (%)
24.28 (12.38-32.52)
23.6 (7.02-38.04)
Total motile sperm count‡ (n x 106) 13.37 (4.11-34.52)
4.86 (1.19-9.17)
8-OHdG‡ (μmol/dL)
10.91 (9.69-13.38)
10.22 (8.52-11.65)
†Wilcoxon rank-sum test.
‡Median values (25th–75th percentile)
P†
0.0249
<0.0001
0.8453
0.0019
0.1853
表 9 低 ORP 群での治療による精液パラメーターの変化。
Parameters
Semen volume§ (mL)
Sperm concentration§
(nx106/mL)
Sperm motility§ (%)
TMC§ (n x106)
Antioxidants (n = 24)
Baseline
3 months
2.35 (1.56-3.33) 2.1 (1.1-3.83)
P†
0.945
Methylcobalamin (n = 20)
Baseline
3 months
1.9 (1.43-3.03) 2 (1.23-4.5)
P†
0.131
P‡
0.571
38.6 (15.6-52.6)
21.5 (7.5-44.4)
0.287
16.6(9.3-28.3)
19 (9.3-27.6)
0.216
0.612
23.8 (11.3-32.9)
21.3 (4.6-45.7)
21.3 (15.9-40.6)
12.6 (4.7-28.7)
0.098
0.419
23.3 (9.7-31.5)
9.0 (2.3-17.5)
11.4 (7.5-32.3)
5.6 (2.9-13.2)
0.870
0.674
0.081
0.207
† Wilcoxon signed-rank sum test.
‡Wilcoxon rank-sum test
§ Median values (25th–75th percentile)
56
表 10 高 ORP 群での治療による精液パラメーターの変化。
Parameters
Semen volume¶ (mL)
Sperm concentration¶
(nx106/mL)
Sperm motility¶ (%)
TMC¶ (n x106)
Antioxidants (n = 12)
Baseline
3 months
2.95 (2.55-4.36) 3.15 (2.15-3.95)
6.1 (4.3-11.3)
12.7 (8.9-26.3)
P‡
0.664
0.043
Methylcobalamin (n = 9†)
Baseline
3 months
P‡
2.7 (1.9-3.62)
2.1 (1.8-3.25)
0.215
5.2 (4.90-12.23) 15.4 (10.3-41.3) 0.008
P§
0.176
0.303
12.0 (6.1-39.0)
4.6 (1.1-5.7)
0.204
0.027
28.7 (12.7-41.4) 16.1 (3.6-48.7)
5.6 (2.3-9.9)
7.4 (1.9-29.8)
0.939
1.000
15.7 (4.4-38.7)
7.6 (2.2-16.5)
0.195
0.641
† The number of samples used for the analysis of sperm motility and TMC were not 9 as
indicated in the table, but 8 instead.
‡Wilcoxon signed-rank sum test.
§ Wilcoxon rank-sum test
表 11 精子濃度が改善した患者、改善しなかった患者における、治療後の ORP(mV/106
sperm/mL)の変化
Improvement of
concentration
Antioxidants
Yes
No
Baseline
Methylcobalamin
3 months
P†
Baseline
2.64 (1.22-12.91)‡ 2.69 (0.72-4.95)‡ 0.109
13
2.74 (1.62-8.10)‡ 4.39 (1.77-6.15)‡ 0.542
1.20 (0.33-1.97)‡
23
1.03 (0.24-2.07)‡ 2.43 (1.37-6.14)‡ 0.029
1.53 (0.81-3.29)‡ 0.049
Abbreviations: ORP, oxidation-reduction potential.
†Wilcoxon signed-rank sum test.
‡Median values (25th–75th percentile)
57
3 months
P†
13
16
...
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