Human granulosa cells in follicular fluids identification
As shown in Figure 1, most of the cells in the dishes were granulosa cells, which were characterized by a positive FSHR staining. Non-specific staining was not detected.
Relationship of the miR-320a-3p and miR-483-5p levels in the granulosa cells and embryo developmental competence
The patients were subdivided into four groups according to the relative expression of miR-320a-3p levels quartile in the granulosa cells: Q1: 0.46-6.17×103, n=49; Q2: 6.41×103-2.35×105, n=49; Q3: 2.63×105-2.34×106, n=49; and Q4: 2.51×106-9.38×107, n=48. The relative expression of miR-483-5p levels quartile: Q1: 0.002-0.18, n=49; Q2: 0.18-1.13, n=49; Q3:1.21-5.80, n=49; and Q4: 5.81-3.52×103, n=48, respectively.
The relative expression of miR-320a-3p levels across different patient groups were significantly different in the good quality embryo rates, with lowest levels in the Q4 intervals (Table 2, P < 0.01). However, the normal fertilized rate for IVF or ICSI and blastocyst rate did not differ (Table 1 and Table 2, P > 0.05).
The relative expression of miR-483-5p levels across different patient groups were no difference (P > 0.05) in the normal fertilization rates for IVF or ICSI, good quality embryo rate, and blastulation rate, as shown in Table 1 and Table 2.
Table 2 Association between the levels of miR-320-3p andmiR-483-5p in granulosa cells from human follicular fluids and embryo developmental competence.
Parameters
|
|
|
|
|
|
|
|
|
P-value
|
|
Q1
|
%
|
Q2
|
%
|
Q3
|
%
|
Q4
|
%
|
|
miR-320a-3p
|
|
|
|
|
|
|
|
|
|
Good quality embryo rate
|
212/296
|
71.6
|
205/275
|
74.5
|
214/296
|
72.3
|
149/262
|
56.9
|
< 0.0001
|
Blastocysts rate
|
81/212
|
38.2
|
65/205
|
31.7
|
82/214
|
38.3
|
55/149
|
36.9
|
NS
|
miR-483-5p
|
|
|
|
|
|
|
|
|
|
Good quality embryo rate
|
225/272
|
82.7
|
203/310
|
65.5
|
197/256
|
77.0
|
155/291
|
53.3
|
NS
|
Blastocysts rate
|
86/225
|
38.2
|
78/203
|
38.4
|
55/197
|
27.9
|
64/155
|
41.3
|
NS
|
Relationship of the miR-320a-3p levels in the granulosa cells and assisted reproductive technology outcomes
The median quantity and the IQR of the miR-320a-3p levels in 195 human granulosa cells samples from pregnancy and unpregnant groups were 2.47×106 (IQR: 6.42×103-1.91×106) and 6.21×106 (IQR: 4.86×103-2.72×106). The median quantity and the IQR of the miR-483-5p levels from pregnancy and unpregnant groups, abortion and non-abortion groups were 44.32 (IQR: 0.19-5.56) and 10.61 (IQR: 0.15-5.28), 4.77 (IQR: 0.06-6.05) and 46.39 (IQR: 0.23-5.20), respectively.
The relative expression levels of miR-320a-3p were associated negetively with clinical pregnancy rate (Table 3, P<0.05). No correlation was observed between the relative expression of miR-320a-3p levels and abortion rate (Table 3, P>0.05). The relative expression of miR-483-5p levels were not associated with clinical pregnancy rate and abortion rate (Table 3, P>0.05).
Table 3 Association between the levels of miR-320-3p and miR-483-5p in granulosa cells from human follicular fluids and reproductive outcomes of assisted reproductive technology.
Parameters
|
|
miR-320a-3p
|
miR-483-5p
|
|
N(%)
|
MD(IQR)
|
95% CI
|
P-value
|
MD(IQR)
|
95% CI
|
P-value
|
Clinical pregnancy rate
|
65.83
|
|
|
0.0477*
|
|
|
NS
|
Pregnancy
|
106 (65.83)
|
2.47×106 (6.42×103-1.91×106)
|
3.79×104,7.43×106
|
|
44.32(0.19-5.56)
|
-2.27×106, 6.48×106
|
|
Unpregnant
|
55 (34.16)
|
6.21×106 (4.86×103-2.72×106)
|
|
|
10.61(0.15-5.28)
|
|
|
Abortion rate
|
5.71
|
|
|
NS
|
|
|
NS
|
Abortion
|
6 (5.71)
|
4.04×105(1.54×104-6.50×105)
|
-126.0,58.57
|
|
4.77(0.06-6.05)
|
-248.10,331.40
|
|
Non-abortion
|
99 (94.29)
|
2.51×106(7.93×103-1.94×106)
|
|
|
46.39(0.23-5.20)
|
|
|
MD Median, IQR interquartile range, CI confidence interval.
Effect of patients’ clinical characteristics on the miR-320a-3p and miR-483-5p levels in the granulosa cells
The relative expression of miR-320a-3p in the granulosa cells was correlated positively with age (β ± SE: 4.79×105 ± 1.61×105, P=0.0033) (Table 4, Figure 2). Moreover, both the basal FSH (β ± SE: 7.90×105 ± 2.14×105, P=0.0003) (Table 4, Figure 2) and ovarian stimulation protocol, including mild stimulation protocol and luteal phase stimulation (β ± SE: 8.27×10-9 ± 2.92×10-9, 6.29×10-9 ± 2.09×10-9, respectively; P=0.006, P=0.004, respectively) (Table 4) significantly and positively affected miR-320a-3p levels in the granulosa cells. The days of stimulation was negatively correlated with the relative expression of miR-320a-3p in the granulosa cells (β ± SE: -6.85×105 ± 3.42×105, P= 0.0466) (Table 4, Figure 2). The relative expression of miR-320a-3p in the granulosa cells was not associated with BMI, basal LH, basal E2, AMH, AFC and total dose of gonadotropins (Table 1, P>0.05).
The relative expression levels of miR-483-5p in the granulosa cells were correlated significantly positively with AMH (β ± SE: 17.55 ± 6.14, P=0.0047) (Table 4, Figure 2). However, miR-483-5p levels were also not correlated with other indicators (Table 4, P>0.05).
Table 4 Patients’ characteristics association with the miR-320a-3p and the miR-483-5p levels in human follicular fluid.
Variable
|
Min-Max
|
Mean
|
n (%)
|
SD
|
miR-320a-3p
|
miR-483-5p
|
|
|
|
|
|
β ± SE
|
P-value
|
β ± SE
|
P-value
|
Age (years)
|
21-46
|
34.39
|
195 (100)
|
5.19
|
4.79×105±1.61×105
|
0.0033*
|
-3.58 ± 3.51
|
0.3084
|
BMI (kg/m2)
|
8.93-32.40
|
22.53
|
194 (99.49)
|
3.39
|
1.45×105 ± 2.54×105
|
0.5678
|
-1.48 ± 5.43
|
0.7850
|
Female baseline levels
|
Basal FSH (IU/L)
|
1.25-33.00
|
8.40
|
195 (100)
|
3.86
|
7.90×105 ± 2.14×105
|
0.0003*
|
-3.42 ± 4.72
|
0.4701
|
Basal LH (IU/L)
|
0.65-40.02
|
4.76
|
195 (100)
|
3.77
|
7.61×103 ± 2.27×105
|
0.9733
|
-0.97 ± 4.85
|
0.8412
|
Basal E2 (pg/ml)
|
2.74-5178
|
76.17
|
195 (100)
|
370.11
|
-6.09×102 ± 2.3×103
|
0.7926
|
-7.57 ×10-4± 4.94×10-2
|
0.9878
|
AMH (ng/ml)
|
0.06-14.62
|
3.52
|
194 (99.49)
|
2.93
|
-1.91×105 ± 2.93×105
|
0.5147
|
17.55 ± 6.14
|
0.0047*
|
Antral follicle count
|
3-52
|
15.74
|
195 (100)
|
7.97
|
-5.39×104 ± 1.07×105
|
0.6159
|
-0.13 ± 2.30
|
0.9535
|
Days of stimulation
|
5-22
|
9.97
|
195 (100)
|
2.48
|
-6.85×105 ± 3.42×105
|
0.0466*
|
0.31 ± 7.38
|
0.9660
|
Total dose of gonadotropins (IU)
|
900-6450
|
2344.27
|
195 (100)
|
842.52
|
-1.39×103 ± 1.01×103
|
0.1704
|
-7.40×10-3 ± 2.17×10-2
|
0.7333
|
Ovarian stimulation protocol
|
Ultra-long protocol
|
-
|
-
|
62 (31.79)
|
-
|
|
|
|
Ref
|
Long protocol
|
-
|
-
|
9 (0.05)
|
-
|
-6.05×10-9 ± 5.36×10-9
|
0.263
|
-2.57×10-5 ± 9.61×10-5
|
0.790
|
Antagonist protocol
|
-
|
-
|
75 (0.38)
|
-
|
-7.10×10-9 ± 6.15×10-9
|
0.250
|
-1.76×10-4 ± 1.42×10-4
|
0.217
|
Progestin-primed ovarian stimulation (PPOS)
|
-
|
-
|
40 (0.21)
|
-
|
2.19×10-9 ± 4.37×10-9
|
0.617
|
-1.30×10-4 ± 1.40×10-4
|
0.354
|
Mild stimulation protocol
|
-
|
-
|
5 (0.03)
|
-
|
8.27×10-9 ± 2.92×10-9
|
0.006*
|
-1.44×10-5 ± 7.61×10-5
|
0.851
|
Luteal phase stimulation
|
-
|
-
|
4 (0.02)
|
-
|
6.29×10-9 ± 2.09×10-9
|
0.004*
|
-1.93×10-5 ± 6.91×10-5
|
0.781
|