From October to November 2019, 44 embryos were included in the study, coming from eight couples who underwent IVF procedures at the Yasmin Clinic, RSCM Kencana, Jakarta.
Table 1
Distribution of Embryos According to Morphological Assessments
Variable
|
|
(n = 44)
|
%
|
Not Blastocyst
|
|
13
|
29,5
|
Early Blast
|
1 AA
|
3
|
6,8
|
|
1 AB
|
2
|
4,5
|
|
1 BB
|
3
|
6,8
|
|
1 CC
|
1
|
2,3
|
Blastocyst
|
2 AA
|
2
|
4,5
|
|
2 CC
|
1
|
2,3
|
|
3 AA
|
1
|
2,3
|
|
3 AB
|
1
|
2,3
|
|
3 BB
|
1
|
2,3
|
Exp Blastocyst
|
4 AA
|
14
|
31,8
|
|
4 AB
|
2
|
4,5
|
Total
|
|
44
|
100
|
Of the 44 embryos that were the subjects of the study, 29.5% did not become blastocysts. Table 1 shows the distribution of embryos according to their morphology. From 70.5% (31), embryos that became blastocysts found 16 embryos with good quality (4 AA and 4 AB).
Embryos in this study were from eight patients. The mean age of patients was 30.6 ± 4.7 years, with the youngest age at 25 years and oldest at 39 years. The average length of infertility is 4.9 ± 5.9 years, with a minimum of 1 year and a maximum of 19 years. Meanwhile, the most common causes of infertility are sperm factor (n = 4), followed by polycystic ovarian syndrome (n = 2), endometriosis (n = 1), and unexplained infertility (n = 1).
Examination by FTIR spectroscopy produces a spectrum of reflectant transmissions derived from molecular vibrations. Figure 1A illustrates the curve between the reflectance values and the wave numbers obtained from 44 samples.
From 44 samples whose spectra are depicted by the colored stripes above, it is found that variations are indeed expected from the embryo metabolism. By calculating the average value of each wave number of groups that are good quality blastocysts and not good quality blastocysts, Fig. 1B is obtained.
Figures 1A and 1B show 44 FTIR spectrum generated from the sample. All spectra show bands almost similar to differences in intensity that are difficult to distinguish visually. There is an absorption peak in several wave numbers, and this is related to various types of functional groups derived from organic compounds in embryo culture medium. Wave number 400–900 cm− 1 is the fingerprint region of each compound (not a functional group region)19; because the culture medium contains many compounds, it is difficult to determine where the compounds are from. At wave number 3000–3500 cm− 1, there is a widening of the peak intensity. This area is related to the functional group strain O-H.19, 20 There is a picture of the absorption peak at wave numbers 1600–1820 cm− 1 in both groups. This area is related to the C = O strain of the carboxylate. Wave numbers 3182–3330 cm− 1 are uptake of carboxylic acids. To connect the small differences in each sample and obtain more appropriate information, processing data was performed.
The distribution patterns of the sample groups were evaluated using PCA by using FTIR spectrum reflectance data in the wave number range 399 to 4000 cm−1. In this analysis, 12 main components were obtained, and it can provide 98% information of the total variance. The plot obtained from principal component shows the pattern found in the sample. The similarity of the sample is marked by the closeness of one sample to another. Figure 2 is a plot score showing the PC1 plot as the x-axis and PC3 as the y-axis. This plot score illustrates that an embryo has become good quality blastocysts and no, cannot be distinguished, although it is starting to be seen in quadrant 2 where there is no good quality blastocyst.
In the chemometric analysis with this PCA, there was still an overlap between the two groups (Fig. 2). This shows that some typical metabolomic patterns are common in both groups, such as the use of pyruvate by embryos. For this reason, the selection of variables has a role in distinguishing if the blastocysts have a good quality or not between groups. With the ReliefF method, the information gain and gain ratio obtained the following wave numbers: 778, 813–831, 1189, 1899–1901, 2659–2665, 3307, and 3367–3398 cm− 1. Wave numbers 700–1000 cm− 1 are the vibration of C = C–H bond and Ar–H bending.19 Meanwhile, wave number 1189 cm− 1 is related to deoxyribose,21 whereas wave numbers 1899–1901 cm− 1 are showing strain at C = O (aldehydes, ketones, amides, esters, and anhydrides).19 Wave numbers 2659–2665 cm− 1 represent the strain on the N–H bond associated with amide. Wave number 3307 cm− 1 corresponds to amide that is the formation of amino acids, and wave numbers 3367–3398 cm− 1 represent the vibrations of O–H, N–H, and C–H.21
Taking into account each wave number that has the role of distinguishing between the two groups, classification analysis used several methods, namely, Tree, SVM, Random Forest, and Logistic Regression. Table 2 shows the best precision and AUC, which is using a prediction model with logistic regression.
Table 2
Evaluation Results of Several Prediction Models in Sample Day − 1
Prediction Models
|
AUC
|
Accuracy
|
Precision
|
Sensitivity
|
Tree
|
0,661
|
0,705
|
0,696
|
0,705
|
SVM
|
0,737
|
0,727
|
0,722
|
0,727
|
Random Forest
|
0,693
|
0,682
|
0,670
|
0,682
|
Logistic Regression
|
0,752
|
0,727
|
0,755
|
0,727
|
AUC, area under the curve, Tree, Decision Tree; SVM, Support Vector Machine.
From the logistic regression prediction model, a receiver operating characteristic (ROC) curve can be obtained as shown in Fig. 3.
Based on the ROC curve, the prediction model has an AUC value of 0.752. This AUC value shows that this prediction model can be used to predict which embryos will become blastocysts or not. Table 3 shows the predictions of the 44 samples of culture medium using the prediction model above.
Table 3
Prediction of good quality blastocyst formation based on FTIR spectrum analysis of day-1 embryo culture medium
Blastocyst
|
Prediction
|
Total
|
Y
|
N
|
|
Actual
|
Y
|
8
|
8
|
16
|
|
N
|
1
|
27
|
28
|
Total
|
|
9
|
35
|
44
|
Based on Table 3, a positive predictive value (PPV) of 88.9% and negative predictive value (NPV) of 77.1% can be obtained.
Table 4
Results of Examination of Culture Medium Compounds using LC-MS / MS
Compound name
|
Apex m/z
|
N-Acetyl –DL-tryptophan
|
247,107
|
Valpromide
|
144,1381
|
3-tert-Butyladipic acid
|
201,1124
|
Indole-3-acrylic acid
|
188,0702
|
Cyclucron
|
199,1802
|
DL-Tryptophan
|
205,0969
|
Azelaic acid
|
187,0966S
|
Valine
|
118,0862
|
L-Norleucine
|
132,1017
|
Triethyl phosphate
|
183,0775
|
N-Acetyl-DL-tryptrophan
|
245,0926
|
Sebacate (decanedionate)
|
201,1124
|
Phenylalanine
|
166,0859
|
Beta-hydroxyisovalorey- L carnitine
|
262,1644
|
5-Oxoproline
|
130,0498
|
N-acetylcitruline
|
218,113
|
Piperidin
|
86,09667
|
5-aminovalerate
|
118,0862
|
Proline
|
116,0706
|
Sphinganin
|
302,3049
|
Inosin
|
269,0888
|
Isoleucin
|
132,1017
|
N-acetylkynurenine
|
251, 1022
|
Based on the LCMS results in Table 4, we can get a picture of the FTIR spectrum of each compound from the spectrum database and then connect it with the wave numbers that play a role in differentiating groups that succeed in becoming good quality blastocysts and not. Then, we found on the first day that the compound that played a role was N-acetyl-Tryptopan. From the FTIR results obtained, the wave numbers that plays a role are 3367–3398 cm− 1, which describe the vibrations of the O–H, N–H, and C–H functional groups. From FTIR, only functional groups are obtained. LCMS/MS compounds above N-acetyl-Tryptopan has an FTIR image with a transmission on these waves.