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Abstract

Context

Late follicular phase elevation in serum progesterone (P) during controlled ovarian hyperstimulation negatively affects the outcome of assisted reproductive technology by contributing to endometrial-embryo asynchrony. There are still no data on lipid metabolite alterations during this process.

Objectives

To investigate alterations in the lipid profile during the window of implantation in patients with premature P rise.

Design

Lipidomic variations in the endometrium were evaluated by ultrahigh-performance liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry.

Setting

University assisted reproductive medicine unit.

Patients or Other Participants

Forty-three patients undergoing in vitro fertilization/intracytoplasmic sperm injection because of a tubal factor or male factor infertility were included in this study. The patients were divided into a high P group (P ≥ 1.5 ng/mL, 15 patients) and a normal P group (P < 1.5 ng/mL, 28 patients) on the day of human chorionic gonadotropin administration.

Interventions

The endometrial tissues were obtained by Pipelle biopsy 7 days after human chorionic gonadotropin administration.

Main Outcome Measures

Alterations in lipid metabolites.

Results

A total of 1026 ions were identified, and 25 lipids were significantly upregulated. The endometrial lipid profile was characterized by substantial increases in the concentrations of phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, diacylglycerol, ceramide, phosphatidylinositol, and phosphatidylserine in patients with a premature P rise at the end of the follicular phase. The correlation analysis between P levels and lipids showed a stronger negative correlation between phosphatidylethanolamine or phosphatidylserine and P levels.

Conclusions

Premature P elevation disrupts the lipid homeostasis of the endometrium during the peri-implantation period. The altered lipid levels may impair endometrial receptivity and early embryo implantation.

Late follicular phase elevation in serum progesterone (P) throughout ovarian stimulation in stimulated in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles is a frequent occurrence. Emerging evidence shows a negative effect on endometrial receptivity and assisted reproductive technology outcomes (1). In stimulated IVF/ICSI cycles, a premature elevation in P may occur in up to 38% of cycles, independent of the stimulation protocol used (2). The current knowledge on the cause of a premature P rise is still limited. However, it seems that supraphysiological hormonal levels resulting from enhanced FSH stimulation during the end of the follicular phase promote a premature P rise (3). The consequence of elevated peripheral P levels is abnormally accelerated endometrial maturation, resulting in impaired endometrial receptivity and asynchrony between the endometrium and embryo (4). Different cutoff values have been used to define an elevated P level during stimulated cycles (5–7), and a significant decrease in ongoing pregnancy rates has been demonstrated with a threshold above 1.5 ng/mL on the day of human chorionic gonadotropin (hCG) administration (8–10). However, the mechanism leading to lower pregnancy rates is unclear. Elevated P levels adversely affect endometrial gene expression and epigenetic modifications (11, 12).

Lipidomics is an emerging tool to predict endometrial receptivity (13). The investigation of molecules that are differentially expressed throughout the “window of implantation” has helped us to identify novel putative markers and guide the clinical acquisition of a receptive endometrium (14). Lipidomics can be defined as the large-scale study of lipid species and their related networks as well as metabolic pathways. Lipids do not belong to a characteristic functional group, and different lipid species are present in diverse substances. Lipidomics has emerged as a novel field to integrate the investigation of the roles of genomics, transcriptomics, proteomics, and metabolomics in cell functions (15).

In this study, we investigated the lipidomics alterations in the endometrium during the peri-implantation period in patients with high P levels on the day of hCG administration during IVF/ICSI cycles. We aimed to provide insight into the mechanism of the premature P elevation in terms of endometrial receptivity.

Materials and methods

Study design and participants

A retrospective cohort study was carried out in the Center of Reproductive Medicine, the Sixth Affiliated Hospital, Sun Yat-sen University, from 2014 to 2016. All of the participants underwent assisted reproduction (IVF or ICSI) because of tubal or male factor infertility. A total of 43 participants were enrolled in the study. The inclusion criteria were as follows: (1) age between 25 and 38 years; (2) regular menstrual cycle (28 to 35 days); (3) normal cycle day 2 to 3 hormone levels [FSH <10 IU/L; LH <10 IU/L, and estradiol (E2) <50 pg/mL]; and (4) body mass index (BMI) between 18 and 28 kg/m2. The exclusion criteria were as follows: (1) endometrial polyps; (2) polycystic ovary syndrome; (3) hydrosalpinx; (4) uterine fibroids; (5) intrauterine adhesions; and (6) adenomyosis. A cutoff level of 1.5 ng/mL was chosen as the definition of an elevation in serum P on the day of hCG administration. This study was approved by the institutional review board of the Sixth Hospital, Sun Yat-sen University, and written informed consent was obtained from all participants (approval number: 2013ZSLYEC-007).

Stimulation protocol and sample collection

All patients underwent assisted reproduction (IVF or ICSI) with controlled ovarian hyperstimulation, via suppression of premature ovulation by GnRH agonists (n = 33) or antagonists (n = 14). Triptorelin depot (IPSEN Biothech, Paris, France) was administered for downregulation, and recombinant FSH (rFSH, Gonal-F; Serono Laboratories, Aubonne, Switzerland) was administered for ovarian stimulation. The dose of gonadotropin was individualized for each patient according to age, antral follicle count, and FSH level on days 2 through 5. Follicle monitoring was carried out routinely. Patients with the antagonist protocol received the GnRH antagonist Cetrorelix (Cetrotide; Merck-Serono, Geneva, Switzerland) starting on day 5 after the onset of stimulation with rFSH. When two or more follicles reached 18 mm in diameter or when more than three follicles reached a mean diameter of 16 to 18 mm, 6000 to 10,000 IU hCG was injected on the day of hCG administration. The specimens were obtained from patients who cancelled their fresh embryo transfer resulting from high P levels (P ≥ 1.5 ng/mL) on the day of hCG administration (n = 15) to avoid the risk of ovarian hyperstimulation syndrome (n = 23) or for personal reasons (n = 5). Our routine practice was to obtain blood samples for sex hormone measurements (H, E2, and P) on the day of hCG administration and on the day of oocyte retrieval. Samples were tested with an electrochemiluminescence system (Roche Diagnostics, Basel, Switzerland). The intra-assay and interassay coefficients of variation were 6.1% and 7.0% for E2, 5.5% and 6.2% for P, 2.8% and 4.5% for FSH, and 1.2% and 2.2% for LH, respectively. All endometrial samples were collected using a Pipelle sampler under sterile conditions 7 days after the hCG injection (hCG + 7) during the window of implantation. After collection, specimens were immediately placed into microtubes and preserved at –80°C until further analysis.

Sample preparation for lipidomic analysis

All of the samples were thawed and then washed with PBS. Lipids were extracted according to the modified MTBE method (16). A total of 400 μL of PBS was added to 20 mg of endometrium. Homogenization was performed using Precellys 24 homogenizer (Bertin, Besancon, France). Then, 200 μL of the homogenate was added to a 1.2 mL mixture of methanol/MTBE/water (4:5:5, v/v/v) and vortexed for 2 minutes. Then, the tubes were incubated on ice for 1 hour and vortexed for 1 minute every 15 minutes. The samples were centrifuged at 2000g for 5 minutes, and 200 μL of supernatant from each sample was transferred to a new tube and dried by nitrogen gas. Samples were dissolved in 500 μL of a mixture of methanol/isopropanol (1:1, v/v) and then centrifuged at 18,000g at 4°C for 5 minutes. Finally, 2 μL of supernatant was injected for ultra-high-performance liquid chromatography with high-resolution mass spectrometry (UHPLC-ESI-HRMS) analysis. The quality control sample was generated by mixing 3 μL of each sample.

UHPLC-ESI-HRMS conditions

As described in our previous reports, the Ascentis Express C18 2.7-μm column (100 mm × 2.1 mm, Sigma-Aldrich, St. Louis, MO) was used for chromatographic separation and lipidomics analysis on a Thermo Scientific Dionex Ultimate 3000 UHPLC system (Thermo Scientific, San Jose, CA). The flow rate was 0.3 mL/min, whereas the column temperature was 45°C. The mobile phases were A, 5% acetonitrile in isopropanol with 10 mM ammonium formate and 0.1% formic acid, and B, 50% water in acetonitrile with 10 mM ammonium formate and 0.1% formic acid (17). The gradient program was as follows: 0 to 0.5 minutes 20% A, 7.5 minutes 50% A, 10 minutes 80% A, 20 to 21.9 minutes 100% A, and 22 to 25 minutes 20% A. Mass spectrometry was performed with a Thermo Scientific Q ExactiveTM benchtop Orbitrap mass spectrometer equipped with heated ESI source in ESI positive and negative modes (Thermo Scientific). The main parameters for tandem mass spectrometry included AGC target 1e5; maximum injection time 65 ms; apex trigger 5 to 10 seconds; dynamic exclusion 10.0 seconds; isolation window 1.2 m/z; and normalized collision energy 25 and 35 eV in positive mode and 20, 30, and 40 eV in negative mode. Ionization conditions were operated at spray voltage 3.5 kV and capillary temperature 300°C (18).

Data analysis

The obtained total ion chromatograms and mass spectra from UHPLC-ESI-HRMS were exported as raw files by Xcalibur (Thermo Scientific). The software we used for lipid identification was LipidSearch (Thermo Scientific), which could help to extract the m/z values, retention times, and peak areas. It could also perform lipid identification from accurate precursor m/z and tandem mass spectrometry raw data with the reference of a large-scale database that contained a large amount of information, including mass chromatogram and mass spectra data, and could also evaluate the matching degrees by four grades (A, B, C, D). Only the metabolites that were A or B levels could be selected for further data analysis. [M + H]+, [M + Na]+, and [M + NH4]+ adduct ions were considered precursor ions in positive ion mode and [M − H]− was considered a precursor ion in negative ion mode.

The samples were first divided into the high P group (HP group; P ≥ 1.5 ng/mL, n = 15) and the normal P group (NP group; P < 1.5 ng/mL, n = 28). Orthogonal projection to latent structures discriminant analysis (OPLS-DA) and S-plots in positive and negative modes were performed by SIMCA-P 13.0 Software (Umetrics, Kinnelon, NJ) between the two groups.

To further rule out the influence of high-level estradiol, samples with high levels of E2 (≥3000 pg/mL) were selected and then divided into high P groups (HEHP group; E2 ≥3000 pg/mL, P ≥ 1.5 ng/mL n = 15) and normal P groups (HENP group; E2 ≥3000 pg/mL, P < 1.5 ng/mL, n = 23). OPLS-DA and S-plots in positive and negative modes were also generated. The Shapiro-Wilk test was used to evaluate the normality of the distribution, and statistical significance was calculated using Student t test and the nonparametric Mann-Whitney U test, with P < 0.05 set as the level of statistical significance. Correlation analysis was performed by Pearson correlation. The statistical test was carried out with SPSS 20.0 software (IBM Analytics).

Results

The characteristics of the participants from the HP group and the NP group are summarized in Table 1. There were no important differences in age, BMI, basal FSH level, LH level, or E2 level between the two groups. The number of days of stimulation and the total dose of rFSH were significantly higher in the study group than in the NP group. The serum E2 level in the study group was significantly higher on the day of hCG administration (6320.12 ± 1419.91 vs. 4644.89 ± 2356.08 pg/mL, P = 0.02721). The P level in the study group on the day of hCG administration was much higher than that in the NP group (1.99 ± 0.37 vs. 0.69 ± 0.27 ng/mL, P < 0.0001), and the result was similar on the day of oocyte retrieval. The patients in the NP group had more good-quality embryos on day 3 than did the patients in the study group (Table 1).

Table 1.

Comparison of Characteristics Between the NP and HP Groups

P Level <1.5P Level ≥1.5P Value
ng/mL (n = 28)ng/mL (n = 15)
Age, y30.32 ± 3.1331.64 ± 3.390.2164
BMI, kg/m221.31 ± 2.6522.25 ± 3.20.3084
Basal FSH, IU/L6.2 ± 1.666.39 ± 1.570.7210
Basal LH, IU/L4.59 ± 2.275 ± 1.530.5330
Basal E2, pg/mL35.1 ± 10.6439.49 ± 17.20.3067
Primary infertility, n13100.205
Secondary infertility, n155
Cause of infertility
 Tubal pathology, n2080.235
 Male factor, n87
Stimulation protocol
 Agonist protocol, n16130.104
 Antagonist protocol, n122
Days of stimulation10.68 ± 1.5912.4 ± 0.830.0003
Total dose of rFSH, IU1656.7 ± 585.912371.67 ± 437.610.0001
E2 (pg/mL) on day of hCG administration4644.89 ± 2356.086166.4 ± 1384.690.0272
P (ng/mL) on day of hCG administration0.69 ± 0.271.99 ± 0.37<0.0001
LH (IU/L) on day of hCG administration2.02 ± 2.120.72 ± 0.380.0240
P (ng/mL) on day of oocyte retrieval14.51 ± 6.7727.43 ± 7.08<0.0001
Fertilization rate, %0.67 ± 0.20.67 ± 0.220.9429
Day 3 good-quality embryo rate, %0.72 ± 0.20.6 ± 0.250.0891
P Level <1.5P Level ≥1.5P Value
ng/mL (n = 28)ng/mL (n = 15)
Age, y30.32 ± 3.1331.64 ± 3.390.2164
BMI, kg/m221.31 ± 2.6522.25 ± 3.20.3084
Basal FSH, IU/L6.2 ± 1.666.39 ± 1.570.7210
Basal LH, IU/L4.59 ± 2.275 ± 1.530.5330
Basal E2, pg/mL35.1 ± 10.6439.49 ± 17.20.3067
Primary infertility, n13100.205
Secondary infertility, n155
Cause of infertility
 Tubal pathology, n2080.235
 Male factor, n87
Stimulation protocol
 Agonist protocol, n16130.104
 Antagonist protocol, n122
Days of stimulation10.68 ± 1.5912.4 ± 0.830.0003
Total dose of rFSH, IU1656.7 ± 585.912371.67 ± 437.610.0001
E2 (pg/mL) on day of hCG administration4644.89 ± 2356.086166.4 ± 1384.690.0272
P (ng/mL) on day of hCG administration0.69 ± 0.271.99 ± 0.37<0.0001
LH (IU/L) on day of hCG administration2.02 ± 2.120.72 ± 0.380.0240
P (ng/mL) on day of oocyte retrieval14.51 ± 6.7727.43 ± 7.08<0.0001
Fertilization rate, %0.67 ± 0.20.67 ± 0.220.9429
Day 3 good-quality embryo rate, %0.72 ± 0.20.6 ± 0.250.0891
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Table 1.

Comparison of Characteristics Between the NP and HP Groups

P Level <1.5P Level ≥1.5P Value
ng/mL (n = 28)ng/mL (n = 15)
Age, y30.32 ± 3.1331.64 ± 3.390.2164
BMI, kg/m221.31 ± 2.6522.25 ± 3.20.3084
Basal FSH, IU/L6.2 ± 1.666.39 ± 1.570.7210
Basal LH, IU/L4.59 ± 2.275 ± 1.530.5330
Basal E2, pg/mL35.1 ± 10.6439.49 ± 17.20.3067
Primary infertility, n13100.205
Secondary infertility, n155
Cause of infertility
 Tubal pathology, n2080.235
 Male factor, n87
Stimulation protocol
 Agonist protocol, n16130.104
 Antagonist protocol, n122
Days of stimulation10.68 ± 1.5912.4 ± 0.830.0003
Total dose of rFSH, IU1656.7 ± 585.912371.67 ± 437.610.0001
E2 (pg/mL) on day of hCG administration4644.89 ± 2356.086166.4 ± 1384.690.0272
P (ng/mL) on day of hCG administration0.69 ± 0.271.99 ± 0.37<0.0001
LH (IU/L) on day of hCG administration2.02 ± 2.120.72 ± 0.380.0240
P (ng/mL) on day of oocyte retrieval14.51 ± 6.7727.43 ± 7.08<0.0001
Fertilization rate, %0.67 ± 0.20.67 ± 0.220.9429
Day 3 good-quality embryo rate, %0.72 ± 0.20.6 ± 0.250.0891
P Level <1.5P Level ≥1.5P Value
ng/mL (n = 28)ng/mL (n = 15)
Age, y30.32 ± 3.1331.64 ± 3.390.2164
BMI, kg/m221.31 ± 2.6522.25 ± 3.20.3084
Basal FSH, IU/L6.2 ± 1.666.39 ± 1.570.7210
Basal LH, IU/L4.59 ± 2.275 ± 1.530.5330
Basal E2, pg/mL35.1 ± 10.6439.49 ± 17.20.3067
Primary infertility, n13100.205
Secondary infertility, n155
Cause of infertility
 Tubal pathology, n2080.235
 Male factor, n87
Stimulation protocol
 Agonist protocol, n16130.104
 Antagonist protocol, n122
Days of stimulation10.68 ± 1.5912.4 ± 0.830.0003
Total dose of rFSH, IU1656.7 ± 585.912371.67 ± 437.610.0001
E2 (pg/mL) on day of hCG administration4644.89 ± 2356.086166.4 ± 1384.690.0272
P (ng/mL) on day of hCG administration0.69 ± 0.271.99 ± 0.37<0.0001
LH (IU/L) on day of hCG administration2.02 ± 2.120.72 ± 0.380.0240
P (ng/mL) on day of oocyte retrieval14.51 ± 6.7727.43 ± 7.08<0.0001
Fertilization rate, %0.67 ± 0.20.67 ± 0.220.9429
Day 3 good-quality embryo rate, %0.72 ± 0.20.6 ± 0.250.0891
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A total of 1026 ions were identified in positive mode, and 583 ions were identified in negative mode. As shown in Fig. 1, the OPLS-DA plots showed a clear separation of samples between the HP and NP groups under both positive (Fig. 1A) and negative modes (Fig. 1B), indicating the distinct lipid profiles of these two groups. Then, the S-plots were generated under positive (Fig. 1C) and negative mode (Fig. 1D) to further clarify the differential lipids that contributed the most to the difference between the two groups by VIP values. The differential lipids were selected by a VIP value >1.0 and then validated at the univariate level by Student t test or the Mann-Whitney U test, with P < 0.05 set as the level of statistical significance. Finally, a total of 25 lipids were selected, including 11 metabolites in positive mode and 14 metabolites in negative mode; the specific information of these lipids is summarized in Table 2. Notably, the levels of all of the differential lipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylcholine (LPC), lysophosphatidyl ethanolamine (LPE), diacylglycerol (DG), ceramide (Cer), phosphatidylinositol (PI), and phosphatidylserine (PS), were significantly higher in the NP group. The relative intensity of each differential lipid is shown in Fig. 2. The correlation analysis between the P level and lipids showed a stronger negative correlation between PE (18:0p/18:1)+H and the P level (r = −0.5235, P = 0.0003), PS (16:0/19:0)−H and the P level (r = −0.6340, P = 0.0063), PE (18:1p/18:1)−H and the P level (r = −0.6060, P = 0.0006), and PE (18:0p/18:1)−H and the P level (r = −0.5003, P = 0.0006) (Table 3).

Figure 1.
OPLS-DA plots of endometrium samples for the HP group (n = 15, gray diamonds) and the NP group (n = 28, black diamonds) under (A) positive mode and (B) negative mode. (C) S-plot under positive mode with all identified differential lipids highlighted in gray. (D) S-plot under negative mode with identified differential lipids highlighted in gray.
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OPLS-DA plots of endometrium samples for the HP group (n = 15, gray diamonds) and the NP group (n = 28, black diamonds) under (A) positive mode and (B) negative mode. (C) S-plot under positive mode with all identified differential lipids highlighted in gray. (D) S-plot under negative mode with identified differential lipids highlighted in gray.

Table 2.

Detailed Information About Significantly Changed Lipids Between the NP and HP Groups

Lipid MoleculeMolecular FormulaAdductm/ztR (min)P ValueVIP Value
Positive mode
 PC(16:0/18:1)C42H82O8N1P1M+H760.583212.95940.038.88
 PC(18:0/18:1)C44H86O8N1P1M+H788.614912.1910.035.30
 PC(16:0e/16:1)C40H80O7N1P1M+H718.573611.79170.021.25
 PE(16:0p/18:1)C39H76O7N1P1M+H702.541412.12420.0032.20
 PE(18:0p/18:1)C41H80O7N1P1M+H730.572812.37340.0091.59
 PE(18:1p/18:1)C41H78O7N1P1M+H728.557412.12290.0191.30
 LPC(18:0)C26H54O7N1P1M+H524.37046.93000.051.48
 LPC(18:1)C26H52O7N1P1M+H522.35485.04200.021.08
 LPE(18:0)C23H48O7N1P1M+H482.32337.13520.011.68
 DG(18:1/18:1)C39H75O5M+NH4638.571012.61070.0451.43
 Cer(d18:1/16:0)C34H67O3N1M+H538.518412.00640.0131.40
Negative mode
 PI(18:0/18:1)C45H85O1N0P1M−H863.563811.76750.0133.49
 PI(16:0/18:1)C43H81O13N0P1M−H835.532511.38750.0022.11
 PI(16:0/20:4)C45H79O13N0P1M−H857.516911.19140.0471.33
 PI(16:0/22:6)C47H79O13N0P1M−H881.517211.05370.0161.11
 PE(18:1p/18:1)C41H78O7N1P1M−H726.543412.12150.0082.56
 PE(16:0/18:1)C41H80O8N1P1M−H744.552511.87510.0282.31
 PE(18:0p/18:1)C41H80O7N1P1M−H728.559612.55810.0092.11
 PE(18:1/20:3)C43H78O8N1P1M−H766.53711.77150.0151.56
 PE(16:0p/18:2)C39H74O7N1P1M−H698.511511.88690.021.24
 PE(16:0/16:1)C39H76O8N1P1M−H716.523611.59500.0211.11
 PE(16:0p/18:1)C39H76O7N1P1M−H700.527912.05300.0063.29
 PS(20:0/18:1)C44H84O10N1P1M−H816.575512.10530.0061.24
 PS(16:0/19:0)C41H80O10N1P1M−H776.544511.67310.0482.58
 LPE(18:0)C23H48O7N1P1M−H480.30867.17600.0042.44
Lipid MoleculeMolecular FormulaAdductm/ztR (min)P ValueVIP Value
Positive mode
 PC(16:0/18:1)C42H82O8N1P1M+H760.583212.95940.038.88
 PC(18:0/18:1)C44H86O8N1P1M+H788.614912.1910.035.30
 PC(16:0e/16:1)C40H80O7N1P1M+H718.573611.79170.021.25
 PE(16:0p/18:1)C39H76O7N1P1M+H702.541412.12420.0032.20
 PE(18:0p/18:1)C41H80O7N1P1M+H730.572812.37340.0091.59
 PE(18:1p/18:1)C41H78O7N1P1M+H728.557412.12290.0191.30
 LPC(18:0)C26H54O7N1P1M+H524.37046.93000.051.48
 LPC(18:1)C26H52O7N1P1M+H522.35485.04200.021.08
 LPE(18:0)C23H48O7N1P1M+H482.32337.13520.011.68
 DG(18:1/18:1)C39H75O5M+NH4638.571012.61070.0451.43
 Cer(d18:1/16:0)C34H67O3N1M+H538.518412.00640.0131.40
Negative mode
 PI(18:0/18:1)C45H85O1N0P1M−H863.563811.76750.0133.49
 PI(16:0/18:1)C43H81O13N0P1M−H835.532511.38750.0022.11
 PI(16:0/20:4)C45H79O13N0P1M−H857.516911.19140.0471.33
 PI(16:0/22:6)C47H79O13N0P1M−H881.517211.05370.0161.11
 PE(18:1p/18:1)C41H78O7N1P1M−H726.543412.12150.0082.56
 PE(16:0/18:1)C41H80O8N1P1M−H744.552511.87510.0282.31
 PE(18:0p/18:1)C41H80O7N1P1M−H728.559612.55810.0092.11
 PE(18:1/20:3)C43H78O8N1P1M−H766.53711.77150.0151.56
 PE(16:0p/18:2)C39H74O7N1P1M−H698.511511.88690.021.24
 PE(16:0/16:1)C39H76O8N1P1M−H716.523611.59500.0211.11
 PE(16:0p/18:1)C39H76O7N1P1M−H700.527912.05300.0063.29
 PS(20:0/18:1)C44H84O10N1P1M−H816.575512.10530.0061.24
 PS(16:0/19:0)C41H80O10N1P1M−H776.544511.67310.0482.58
 LPE(18:0)C23H48O7N1P1M−H480.30867.17600.0042.44

Abbreviations: tR, retention of time; VIP, variable importance in projection.

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Table 2.

Detailed Information About Significantly Changed Lipids Between the NP and HP Groups

Lipid MoleculeMolecular FormulaAdductm/ztR (min)P ValueVIP Value
Positive mode
 PC(16:0/18:1)C42H82O8N1P1M+H760.583212.95940.038.88
 PC(18:0/18:1)C44H86O8N1P1M+H788.614912.1910.035.30
 PC(16:0e/16:1)C40H80O7N1P1M+H718.573611.79170.021.25
 PE(16:0p/18:1)C39H76O7N1P1M+H702.541412.12420.0032.20
 PE(18:0p/18:1)C41H80O7N1P1M+H730.572812.37340.0091.59
 PE(18:1p/18:1)C41H78O7N1P1M+H728.557412.12290.0191.30
 LPC(18:0)C26H54O7N1P1M+H524.37046.93000.051.48
 LPC(18:1)C26H52O7N1P1M+H522.35485.04200.021.08
 LPE(18:0)C23H48O7N1P1M+H482.32337.13520.011.68
 DG(18:1/18:1)C39H75O5M+NH4638.571012.61070.0451.43
 Cer(d18:1/16:0)C34H67O3N1M+H538.518412.00640.0131.40
Negative mode
 PI(18:0/18:1)C45H85O1N0P1M−H863.563811.76750.0133.49
 PI(16:0/18:1)C43H81O13N0P1M−H835.532511.38750.0022.11
 PI(16:0/20:4)C45H79O13N0P1M−H857.516911.19140.0471.33
 PI(16:0/22:6)C47H79O13N0P1M−H881.517211.05370.0161.11
 PE(18:1p/18:1)C41H78O7N1P1M−H726.543412.12150.0082.56
 PE(16:0/18:1)C41H80O8N1P1M−H744.552511.87510.0282.31
 PE(18:0p/18:1)C41H80O7N1P1M−H728.559612.55810.0092.11
 PE(18:1/20:3)C43H78O8N1P1M−H766.53711.77150.0151.56
 PE(16:0p/18:2)C39H74O7N1P1M−H698.511511.88690.021.24
 PE(16:0/16:1)C39H76O8N1P1M−H716.523611.59500.0211.11
 PE(16:0p/18:1)C39H76O7N1P1M−H700.527912.05300.0063.29
 PS(20:0/18:1)C44H84O10N1P1M−H816.575512.10530.0061.24
 PS(16:0/19:0)C41H80O10N1P1M−H776.544511.67310.0482.58
 LPE(18:0)C23H48O7N1P1M−H480.30867.17600.0042.44
Lipid MoleculeMolecular FormulaAdductm/ztR (min)P ValueVIP Value
Positive mode
 PC(16:0/18:1)C42H82O8N1P1M+H760.583212.95940.038.88
 PC(18:0/18:1)C44H86O8N1P1M+H788.614912.1910.035.30
 PC(16:0e/16:1)C40H80O7N1P1M+H718.573611.79170.021.25
 PE(16:0p/18:1)C39H76O7N1P1M+H702.541412.12420.0032.20
 PE(18:0p/18:1)C41H80O7N1P1M+H730.572812.37340.0091.59
 PE(18:1p/18:1)C41H78O7N1P1M+H728.557412.12290.0191.30
 LPC(18:0)C26H54O7N1P1M+H524.37046.93000.051.48
 LPC(18:1)C26H52O7N1P1M+H522.35485.04200.021.08
 LPE(18:0)C23H48O7N1P1M+H482.32337.13520.011.68
 DG(18:1/18:1)C39H75O5M+NH4638.571012.61070.0451.43
 Cer(d18:1/16:0)C34H67O3N1M+H538.518412.00640.0131.40
Negative mode
 PI(18:0/18:1)C45H85O1N0P1M−H863.563811.76750.0133.49
 PI(16:0/18:1)C43H81O13N0P1M−H835.532511.38750.0022.11
 PI(16:0/20:4)C45H79O13N0P1M−H857.516911.19140.0471.33
 PI(16:0/22:6)C47H79O13N0P1M−H881.517211.05370.0161.11
 PE(18:1p/18:1)C41H78O7N1P1M−H726.543412.12150.0082.56
 PE(16:0/18:1)C41H80O8N1P1M−H744.552511.87510.0282.31
 PE(18:0p/18:1)C41H80O7N1P1M−H728.559612.55810.0092.11
 PE(18:1/20:3)C43H78O8N1P1M−H766.53711.77150.0151.56
 PE(16:0p/18:2)C39H74O7N1P1M−H698.511511.88690.021.24
 PE(16:0/16:1)C39H76O8N1P1M−H716.523611.59500.0211.11
 PE(16:0p/18:1)C39H76O7N1P1M−H700.527912.05300.0063.29
 PS(20:0/18:1)C44H84O10N1P1M−H816.575512.10530.0061.24
 PS(16:0/19:0)C41H80O10N1P1M−H776.544511.67310.0482.58
 LPE(18:0)C23H48O7N1P1M−H480.30867.17600.0042.44

Abbreviations: tR, retention of time; VIP, variable importance in projection.

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Figure 2.
Content comparison of differential lipids in the NP and HP groups. Data are represented as the mean ± SD; n = 15 in the HP group, and n = 28 in the NP group. (*P < 0.05; **P < 0.01; vs the NP group.)
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Content comparison of differential lipids in the NP and HP groups. Data are represented as the mean ± SD; n = 15 in the HP group, and n = 28 in the NP group. (*P < 0.05; **P < 0.01; vs the NP group.)

Table 3.

Correlation Analysis Between Progesterone Levels and Lipids in Patients With a Premature Progesterone Rise

Lipid MoleculerP Value
PC(16:0/18:1)+H−0.37630.0129
PC(18:0/18:1)+H−0.40710.0067
LPE(18:0)+H−0.47820.0021
PE(18:0p/18:1)+H−0.52350.0003
LPC(18:0)+H−0.33690.0271
DG(18:1/18:1)+NH4−0.43000.0099
Cer(d18:1/16:0)+H−0.38560.0117
PE(18:1p/18:1)+H−0.48190.0109
PC(16:0e/16:1)+H−0.15870.3551
LPC(18:1)+H−0.37990.0120
PE(16:0p/18:1)+H−0.45950.0019
PI(18:0/18:1)−H−0.43330.0047
PE(16:0p/18:1)−H−0.45260.0023
PS(16:0/19:0)−H−0.63400.0063
PE(18:1p/18:1)−H−0.60600.0006
LPE(18:0)−H−0.48750.0009
PE(16:0/18:1)−H−0.38730.0103
PE(18:0p/18:1)-H−0.50030.0006
PI(16:0/18:1)−H−0.49170.0013
PE(18:1/20:3)−H−0.43820.0222
PI(16:0/20:4)−H−0.35320.0202
PS(20:0/18:1)−H−0.40170.0112
PE(16:0p/18:2)−H−0.45650.0021
PE(16:0/16:1)−H−0.46960.0088
PI(16:0/22:6)−H−0.42280.0066
Lipid MoleculerP Value
PC(16:0/18:1)+H−0.37630.0129
PC(18:0/18:1)+H−0.40710.0067
LPE(18:0)+H−0.47820.0021
PE(18:0p/18:1)+H−0.52350.0003
LPC(18:0)+H−0.33690.0271
DG(18:1/18:1)+NH4−0.43000.0099
Cer(d18:1/16:0)+H−0.38560.0117
PE(18:1p/18:1)+H−0.48190.0109
PC(16:0e/16:1)+H−0.15870.3551
LPC(18:1)+H−0.37990.0120
PE(16:0p/18:1)+H−0.45950.0019
PI(18:0/18:1)−H−0.43330.0047
PE(16:0p/18:1)−H−0.45260.0023
PS(16:0/19:0)−H−0.63400.0063
PE(18:1p/18:1)−H−0.60600.0006
LPE(18:0)−H−0.48750.0009
PE(16:0/18:1)−H−0.38730.0103
PE(18:0p/18:1)-H−0.50030.0006
PI(16:0/18:1)−H−0.49170.0013
PE(18:1/20:3)−H−0.43820.0222
PI(16:0/20:4)−H−0.35320.0202
PS(20:0/18:1)−H−0.40170.0112
PE(16:0p/18:2)−H−0.45650.0021
PE(16:0/16:1)−H−0.46960.0088
PI(16:0/22:6)−H−0.42280.0066

Boldface type indicates r < –0.5.

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Table 3.

Correlation Analysis Between Progesterone Levels and Lipids in Patients With a Premature Progesterone Rise

Lipid MoleculerP Value
PC(16:0/18:1)+H−0.37630.0129
PC(18:0/18:1)+H−0.40710.0067
LPE(18:0)+H−0.47820.0021
PE(18:0p/18:1)+H−0.52350.0003
LPC(18:0)+H−0.33690.0271
DG(18:1/18:1)+NH4−0.43000.0099
Cer(d18:1/16:0)+H−0.38560.0117
PE(18:1p/18:1)+H−0.48190.0109
PC(16:0e/16:1)+H−0.15870.3551
LPC(18:1)+H−0.37990.0120
PE(16:0p/18:1)+H−0.45950.0019
PI(18:0/18:1)−H−0.43330.0047
PE(16:0p/18:1)−H−0.45260.0023
PS(16:0/19:0)−H−0.63400.0063
PE(18:1p/18:1)−H−0.60600.0006
LPE(18:0)−H−0.48750.0009
PE(16:0/18:1)−H−0.38730.0103
PE(18:0p/18:1)-H−0.50030.0006
PI(16:0/18:1)−H−0.49170.0013
PE(18:1/20:3)−H−0.43820.0222
PI(16:0/20:4)−H−0.35320.0202
PS(20:0/18:1)−H−0.40170.0112
PE(16:0p/18:2)−H−0.45650.0021
PE(16:0/16:1)−H−0.46960.0088
PI(16:0/22:6)−H−0.42280.0066
Lipid MoleculerP Value
PC(16:0/18:1)+H−0.37630.0129
PC(18:0/18:1)+H−0.40710.0067
LPE(18:0)+H−0.47820.0021
PE(18:0p/18:1)+H−0.52350.0003
LPC(18:0)+H−0.33690.0271
DG(18:1/18:1)+NH4−0.43000.0099
Cer(d18:1/16:0)+H−0.38560.0117
PE(18:1p/18:1)+H−0.48190.0109
PC(16:0e/16:1)+H−0.15870.3551
LPC(18:1)+H−0.37990.0120
PE(16:0p/18:1)+H−0.45950.0019
PI(18:0/18:1)−H−0.43330.0047
PE(16:0p/18:1)−H−0.45260.0023
PS(16:0/19:0)−H−0.63400.0063
PE(18:1p/18:1)−H−0.60600.0006
LPE(18:0)−H−0.48750.0009
PE(16:0/18:1)−H−0.38730.0103
PE(18:0p/18:1)-H−0.50030.0006
PI(16:0/18:1)−H−0.49170.0013
PE(18:1/20:3)−H−0.43820.0222
PI(16:0/20:4)−H−0.35320.0202
PS(20:0/18:1)−H−0.40170.0112
PE(16:0p/18:2)−H−0.45650.0021
PE(16:0/16:1)−H−0.46960.0088
PI(16:0/22:6)−H−0.42280.0066

Boldface type indicates r < –0.5.

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To avoid the influence of inconsistent estrogen levels on the metabolic spectrum of the endometrium, we further screened lipids at high-level E2 (E2 ≥3000 pg/mL) or low-level E2 (E2 <3000 pg/mL) on the day of hCG administration. Because of the small sample size, we failed to analyze and obtain reliable results for the two groups at low estrogen levels. The OPLS-DA analysis was also performed and indicated a clear separation between the two groups (HEHP and HENP) among patients with E2 ≥3000 pg/mL on the hCG day (Fig. 3A-B). The characteristics of the participants from the HEHP group and the HENP group are summarized in Table 4. Additionally, the S-plots were also generated with the differential lipids highlighted in red (Fig. 3C-D). Finally, a total of 24 lipids were selected, including 11 metabolites in the positive mode and 13 metabolites in the negative mode, which are summarized in Table 5. The levels of these lipids, including PC, PE, LPC, LPE, DG, Cer, PI, and PS, most of which were the same as the differential lipids between the HP and NP groups, were significantly higher in the HENP group, whereas the levels of TG were higher in the HEHP group (Fig. 4). The correlation analysis between the P levels and lipids showed a stronger negative correlation only between PS (16:0/19:0)−H and the P levels (r = −0.6527, P = 0.0083) and PE (18:1p/18:1)−H and the P levels (r = −0.5697, P = 0.0045) (Table 6).

Figure 3.
OPLS-DA plots of samples in the HEHP group (n = 15, gray diamonds) and the HENP group (n = 23, black diamonds) under (A) positive mode and (B) negative mode. (C) S-plot under positive mode with all identified differential lipids highlighted in gray. (D) S-plot under negative mode with identified differential lipids highlighted in gray.
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OPLS-DA plots of samples in the HEHP group (n = 15, gray diamonds) and the HENP group (n = 23, black diamonds) under (A) positive mode and (B) negative mode. (C) S-plot under positive mode with all identified differential lipids highlighted in gray. (D) S-plot under negative mode with identified differential lipids highlighted in gray.

Table 4.

Comparison of Characteristics Between the HENP and HEHP Groups

P Level <1.5P Level ≥1.5P Value
ng/mL (n = 23)ng/mL (n = 15)
Age, y30.09 ± 2.7131.64 ± 3.390.1326
BMI, kg/m221.27 ± 2.3722.25 ± 3.20.2811
Basal FSH, IU/L6.38 ± 1.666.39 ± 1.570.9845
Basal LH, IU/L4.84 ± 2.265 ± 1.530.8089
Basal E2, pg/mL37.4 ± 9.9139.49 ± 17.20.6367
Primary infertility, n9100.097
Secondary infertility, n145
Cause of infertility
 Tubal pathology, n1880.106
 Male factor, n57
Stimulation protocol
 Agonist protocol, n15130.275
 Antagonist protocol, n82
 Days of stimulation10.78 ± 1.6512.4 ± 0.830.0012
Total dose of rFSH, IU1671.2 ± 569.992371.67 ± 437.610.0003
E2 (pg/mL) on day of hCG administration5307.7 ± 2032.256166.4 ± 1384.690.1610
P (ng/mL) on day of hCG administration0.71 ± 0.251.99 ± 0.37<0.0001
LH (IU/L) on day of hCG administration2.02 ± 2.230.72 ± 0.380.0240
P (ng/mL) on day of oocyte retrieval15.21 ± 6.4227.43 ± 7.08<0.0001
Fertilization rate, %0.65 ± 0.190.67 ± 0.220.7279
Day 3 good embryo rate, %0.71 ± 0.190.6 ± 0.250.1173
P Level <1.5P Level ≥1.5P Value
ng/mL (n = 23)ng/mL (n = 15)
Age, y30.09 ± 2.7131.64 ± 3.390.1326
BMI, kg/m221.27 ± 2.3722.25 ± 3.20.2811
Basal FSH, IU/L6.38 ± 1.666.39 ± 1.570.9845
Basal LH, IU/L4.84 ± 2.265 ± 1.530.8089
Basal E2, pg/mL37.4 ± 9.9139.49 ± 17.20.6367
Primary infertility, n9100.097
Secondary infertility, n145
Cause of infertility
 Tubal pathology, n1880.106
 Male factor, n57
Stimulation protocol
 Agonist protocol, n15130.275
 Antagonist protocol, n82
 Days of stimulation10.78 ± 1.6512.4 ± 0.830.0012
Total dose of rFSH, IU1671.2 ± 569.992371.67 ± 437.610.0003
E2 (pg/mL) on day of hCG administration5307.7 ± 2032.256166.4 ± 1384.690.1610
P (ng/mL) on day of hCG administration0.71 ± 0.251.99 ± 0.37<0.0001
LH (IU/L) on day of hCG administration2.02 ± 2.230.72 ± 0.380.0240
P (ng/mL) on day of oocyte retrieval15.21 ± 6.4227.43 ± 7.08<0.0001
Fertilization rate, %0.65 ± 0.190.67 ± 0.220.7279
Day 3 good embryo rate, %0.71 ± 0.190.6 ± 0.250.1173
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Table 4.

Comparison of Characteristics Between the HENP and HEHP Groups

P Level <1.5P Level ≥1.5P Value
ng/mL (n = 23)ng/mL (n = 15)
Age, y30.09 ± 2.7131.64 ± 3.390.1326
BMI, kg/m221.27 ± 2.3722.25 ± 3.20.2811
Basal FSH, IU/L6.38 ± 1.666.39 ± 1.570.9845
Basal LH, IU/L4.84 ± 2.265 ± 1.530.8089
Basal E2, pg/mL37.4 ± 9.9139.49 ± 17.20.6367
Primary infertility, n9100.097
Secondary infertility, n145
Cause of infertility
 Tubal pathology, n1880.106
 Male factor, n57
Stimulation protocol
 Agonist protocol, n15130.275
 Antagonist protocol, n82
 Days of stimulation10.78 ± 1.6512.4 ± 0.830.0012
Total dose of rFSH, IU1671.2 ± 569.992371.67 ± 437.610.0003
E2 (pg/mL) on day of hCG administration5307.7 ± 2032.256166.4 ± 1384.690.1610
P (ng/mL) on day of hCG administration0.71 ± 0.251.99 ± 0.37<0.0001
LH (IU/L) on day of hCG administration2.02 ± 2.230.72 ± 0.380.0240
P (ng/mL) on day of oocyte retrieval15.21 ± 6.4227.43 ± 7.08<0.0001
Fertilization rate, %0.65 ± 0.190.67 ± 0.220.7279
Day 3 good embryo rate, %0.71 ± 0.190.6 ± 0.250.1173
P Level <1.5P Level ≥1.5P Value
ng/mL (n = 23)ng/mL (n = 15)
Age, y30.09 ± 2.7131.64 ± 3.390.1326
BMI, kg/m221.27 ± 2.3722.25 ± 3.20.2811
Basal FSH, IU/L6.38 ± 1.666.39 ± 1.570.9845
Basal LH, IU/L4.84 ± 2.265 ± 1.530.8089
Basal E2, pg/mL37.4 ± 9.9139.49 ± 17.20.6367
Primary infertility, n9100.097
Secondary infertility, n145
Cause of infertility
 Tubal pathology, n1880.106
 Male factor, n57
Stimulation protocol
 Agonist protocol, n15130.275
 Antagonist protocol, n82
 Days of stimulation10.78 ± 1.6512.4 ± 0.830.0012
Total dose of rFSH, IU1671.2 ± 569.992371.67 ± 437.610.0003
E2 (pg/mL) on day of hCG administration5307.7 ± 2032.256166.4 ± 1384.690.1610
P (ng/mL) on day of hCG administration0.71 ± 0.251.99 ± 0.37<0.0001
LH (IU/L) on day of hCG administration2.02 ± 2.230.72 ± 0.380.0240
P (ng/mL) on day of oocyte retrieval15.21 ± 6.4227.43 ± 7.08<0.0001
Fertilization rate, %0.65 ± 0.190.67 ± 0.220.7279
Day 3 good embryo rate, %0.71 ± 0.190.6 ± 0.250.1173
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Table 5.

Detailed Information About Significantly Changed Lipids Between the HEHP and HENP Groups

Lipid MoleculeMolecular FormulaAdductm/ztR(min)P ValueVIP Value
Positive mode
 PC(16:0/18:1)C42H82O8N1P1M+H760.583212.95940.038.42
 PC(18:0/18:1)C44H86O8N1P1M+H788.614912.1910.0215.38
 PC(20:1/18:2)C46H86O8N1P1M+H812.614712.02370.0444.60
 PE(16:0p/18:1)C39H76O7N1P1M+H702.541412.12420.0442.07
 PE(18:0p/18:1)C41H80O7N1P1M+H730.572812.37340.0161.49
 PE(18:1p/18:1)C41H78O7N1P1M+H728.557412.12290.0241.14
 LPC(18:1)C26H52O7N1P1M+H522.35485.04200.0191.12
 LPE(18:0)C23H48O7N1P1M+H482.32337.13520.0141.57
 TG(16:0/18:2/18:3)C55H96O6M+Na875.707814.95680.0211.14
 Cer(d18:1/16:0)C34H68O3N1M+H538.518412.00640.0161.41
 SM(d16:0/18:1)C39H79O6N2P1M+H703.573511.49800.0415.46
Negative mode
 PI(18:0/18:1)C45H85O1N0P1M−H863.563811.76750.013.49
 PI(16:0/18:1)C43H81O13N0P1M−H835.532511.38750.0022.27
 PI(16:0/22:6)C47H79O13N0P1M−H881.517211.05370.0161.14
 PE(18:1p/18:1)C41H78O7N1P1M−H726.543412.12150.0192.04
 PE(16:0/18:1)C41H80O8N1P1M−H744.552511.87510.0242.35
 PE(18:0p/18:1)C41H80O7N1P1M−H728.559612.55810.0162.04
 PE(18:1/20:3)C43H78O8N1P1M−H766.53711.77150.0191.95
 PE(16:0/16:1)C39H76O8N1P1M−H716.523611.59500.0391.16
 PE(16:0p/18:1)C39H76O7N1P1M−H700.527912.05300.0073.14
 PS(16:0/19:0)C41H80O10N1P1M−H776.544511.67310.0282.82
 LPE(18:0)C23H48O7N1P1M−H480.30867.17600.0072.33
 LPE(16:0p)C21H44O6N1P1M−H436.28255.69060.0462.04
 LPE(18:1)C23H46O7N1P1M−H478.29335.34380.0161.01
Lipid MoleculeMolecular FormulaAdductm/ztR(min)P ValueVIP Value
Positive mode
 PC(16:0/18:1)C42H82O8N1P1M+H760.583212.95940.038.42
 PC(18:0/18:1)C44H86O8N1P1M+H788.614912.1910.0215.38
 PC(20:1/18:2)C46H86O8N1P1M+H812.614712.02370.0444.60
 PE(16:0p/18:1)C39H76O7N1P1M+H702.541412.12420.0442.07
 PE(18:0p/18:1)C41H80O7N1P1M+H730.572812.37340.0161.49
 PE(18:1p/18:1)C41H78O7N1P1M+H728.557412.12290.0241.14
 LPC(18:1)C26H52O7N1P1M+H522.35485.04200.0191.12
 LPE(18:0)C23H48O7N1P1M+H482.32337.13520.0141.57
 TG(16:0/18:2/18:3)C55H96O6M+Na875.707814.95680.0211.14
 Cer(d18:1/16:0)C34H68O3N1M+H538.518412.00640.0161.41
 SM(d16:0/18:1)C39H79O6N2P1M+H703.573511.49800.0415.46
Negative mode
 PI(18:0/18:1)C45H85O1N0P1M−H863.563811.76750.013.49
 PI(16:0/18:1)C43H81O13N0P1M−H835.532511.38750.0022.27
 PI(16:0/22:6)C47H79O13N0P1M−H881.517211.05370.0161.14
 PE(18:1p/18:1)C41H78O7N1P1M−H726.543412.12150.0192.04
 PE(16:0/18:1)C41H80O8N1P1M−H744.552511.87510.0242.35
 PE(18:0p/18:1)C41H80O7N1P1M−H728.559612.55810.0162.04
 PE(18:1/20:3)C43H78O8N1P1M−H766.53711.77150.0191.95
 PE(16:0/16:1)C39H76O8N1P1M−H716.523611.59500.0391.16
 PE(16:0p/18:1)C39H76O7N1P1M−H700.527912.05300.0073.14
 PS(16:0/19:0)C41H80O10N1P1M−H776.544511.67310.0282.82
 LPE(18:0)C23H48O7N1P1M−H480.30867.17600.0072.33
 LPE(16:0p)C21H44O6N1P1M−H436.28255.69060.0462.04
 LPE(18:1)C23H46O7N1P1M−H478.29335.34380.0161.01

Abbreviations: tR, retention of time; VIP, variable importance in projection.

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Table 5.

Detailed Information About Significantly Changed Lipids Between the HEHP and HENP Groups

Lipid MoleculeMolecular FormulaAdductm/ztR(min)P ValueVIP Value
Positive mode
 PC(16:0/18:1)C42H82O8N1P1M+H760.583212.95940.038.42
 PC(18:0/18:1)C44H86O8N1P1M+H788.614912.1910.0215.38
 PC(20:1/18:2)C46H86O8N1P1M+H812.614712.02370.0444.60
 PE(16:0p/18:1)C39H76O7N1P1M+H702.541412.12420.0442.07
 PE(18:0p/18:1)C41H80O7N1P1M+H730.572812.37340.0161.49
 PE(18:1p/18:1)C41H78O7N1P1M+H728.557412.12290.0241.14
 LPC(18:1)C26H52O7N1P1M+H522.35485.04200.0191.12
 LPE(18:0)C23H48O7N1P1M+H482.32337.13520.0141.57
 TG(16:0/18:2/18:3)C55H96O6M+Na875.707814.95680.0211.14
 Cer(d18:1/16:0)C34H68O3N1M+H538.518412.00640.0161.41
 SM(d16:0/18:1)C39H79O6N2P1M+H703.573511.49800.0415.46
Negative mode
 PI(18:0/18:1)C45H85O1N0P1M−H863.563811.76750.013.49
 PI(16:0/18:1)C43H81O13N0P1M−H835.532511.38750.0022.27
 PI(16:0/22:6)C47H79O13N0P1M−H881.517211.05370.0161.14
 PE(18:1p/18:1)C41H78O7N1P1M−H726.543412.12150.0192.04
 PE(16:0/18:1)C41H80O8N1P1M−H744.552511.87510.0242.35
 PE(18:0p/18:1)C41H80O7N1P1M−H728.559612.55810.0162.04
 PE(18:1/20:3)C43H78O8N1P1M−H766.53711.77150.0191.95
 PE(16:0/16:1)C39H76O8N1P1M−H716.523611.59500.0391.16
 PE(16:0p/18:1)C39H76O7N1P1M−H700.527912.05300.0073.14
 PS(16:0/19:0)C41H80O10N1P1M−H776.544511.67310.0282.82
 LPE(18:0)C23H48O7N1P1M−H480.30867.17600.0072.33
 LPE(16:0p)C21H44O6N1P1M−H436.28255.69060.0462.04
 LPE(18:1)C23H46O7N1P1M−H478.29335.34380.0161.01
Lipid MoleculeMolecular FormulaAdductm/ztR(min)P ValueVIP Value
Positive mode
 PC(16:0/18:1)C42H82O8N1P1M+H760.583212.95940.038.42
 PC(18:0/18:1)C44H86O8N1P1M+H788.614912.1910.0215.38
 PC(20:1/18:2)C46H86O8N1P1M+H812.614712.02370.0444.60
 PE(16:0p/18:1)C39H76O7N1P1M+H702.541412.12420.0442.07
 PE(18:0p/18:1)C41H80O7N1P1M+H730.572812.37340.0161.49
 PE(18:1p/18:1)C41H78O7N1P1M+H728.557412.12290.0241.14
 LPC(18:1)C26H52O7N1P1M+H522.35485.04200.0191.12
 LPE(18:0)C23H48O7N1P1M+H482.32337.13520.0141.57
 TG(16:0/18:2/18:3)C55H96O6M+Na875.707814.95680.0211.14
 Cer(d18:1/16:0)C34H68O3N1M+H538.518412.00640.0161.41
 SM(d16:0/18:1)C39H79O6N2P1M+H703.573511.49800.0415.46
Negative mode
 PI(18:0/18:1)C45H85O1N0P1M−H863.563811.76750.013.49
 PI(16:0/18:1)C43H81O13N0P1M−H835.532511.38750.0022.27
 PI(16:0/22:6)C47H79O13N0P1M−H881.517211.05370.0161.14
 PE(18:1p/18:1)C41H78O7N1P1M−H726.543412.12150.0192.04
 PE(16:0/18:1)C41H80O8N1P1M−H744.552511.87510.0242.35
 PE(18:0p/18:1)C41H80O7N1P1M−H728.559612.55810.0162.04
 PE(18:1/20:3)C43H78O8N1P1M−H766.53711.77150.0191.95
 PE(16:0/16:1)C39H76O8N1P1M−H716.523611.59500.0391.16
 PE(16:0p/18:1)C39H76O7N1P1M−H700.527912.05300.0073.14
 PS(16:0/19:0)C41H80O10N1P1M−H776.544511.67310.0282.82
 LPE(18:0)C23H48O7N1P1M−H480.30867.17600.0072.33
 LPE(16:0p)C21H44O6N1P1M−H436.28255.69060.0462.04
 LPE(18:1)C23H46O7N1P1M−H478.29335.34380.0161.01

Abbreviations: tR, retention of time; VIP, variable importance in projection.

Open in new tab
Figure 4.
Content comparison of differential lipids in the HEHP and HENP groups. Data are represented as the mean ± SD; n = 15 in the HEHP group, and n = 23 in the HENP group (*P < 0.05; **P < 0.01; **** P < 0.0001 vs the HENP group.).
Open in new tabDownload slide

Content comparison of differential lipids in the HEHP and HENP groups. Data are represented as the mean ± SD; n = 15 in the HEHP group, and n = 23 in the HENP group (*P < 0.05; **P < 0.01; **** P < 0.0001 vs the HENP group.).

Table 6.

Correlation Analysis Between Progesterone Levels and Lipids in Patients With a Premature Progesterone Rise and High Level of E2 (E2 ≥3000 pg/mL) at the End of the Follicular Phase

Lipid MoleculerP Value
PC(16:0/18:1)+H−0.35300.0297
SM(d16:0/18:1)+H−0.34990.0313
PC(18:0/18:1)+H−0.40670.0113
PC(20:1/18:2)+H−0.35440.0339
PE(16:0p/18:1)+H−0.44060.0056
LPE(18:0)+H−0.45990.0062
PE(18:0p/18:1)+H−0.49140.0017
Cer(d18:1/16:0)+H−0.39610.0152
TG(16:0/18:2/18:3)+NH40.33990.0368
PE(18:1p/18:1)+H−0.45420.0295
LPC(18:1)+H−0.40010.0128
PI(18:0/18:1)−H−0.42850.0091
PE(16:0p/18:1)−H−0.42750.0074
PS(16:0/19:0)−H−0.65270.0083
PE(16:0/18:1)−H−0.37650.0198
LPE(18:0)−H−0.45410.0042
PI(16:0/18:1)−H−0.48230.0029
LPE(16:0p)−H−0.36740.0386
PE(18:1p/18:1)−H−0.56970.0045
PE(18:0p/18:1)−H−0.46620.0032
PE(18:1/20:3)−H−0.43490.0298
PE(16:0/16:1)−H−0.45580.0169
PI(16:0/22:6)−H−0.43250.0095
LPE(18:1)−H−0.46920.0030
Lipid MoleculerP Value
PC(16:0/18:1)+H−0.35300.0297
SM(d16:0/18:1)+H−0.34990.0313
PC(18:0/18:1)+H−0.40670.0113
PC(20:1/18:2)+H−0.35440.0339
PE(16:0p/18:1)+H−0.44060.0056
LPE(18:0)+H−0.45990.0062
PE(18:0p/18:1)+H−0.49140.0017
Cer(d18:1/16:0)+H−0.39610.0152
TG(16:0/18:2/18:3)+NH40.33990.0368
PE(18:1p/18:1)+H−0.45420.0295
LPC(18:1)+H−0.40010.0128
PI(18:0/18:1)−H−0.42850.0091
PE(16:0p/18:1)−H−0.42750.0074
PS(16:0/19:0)−H−0.65270.0083
PE(16:0/18:1)−H−0.37650.0198
LPE(18:0)−H−0.45410.0042
PI(16:0/18:1)−H−0.48230.0029
LPE(16:0p)−H−0.36740.0386
PE(18:1p/18:1)−H−0.56970.0045
PE(18:0p/18:1)−H−0.46620.0032
PE(18:1/20:3)−H−0.43490.0298
PE(16:0/16:1)−H−0.45580.0169
PI(16:0/22:6)−H−0.43250.0095
LPE(18:1)−H−0.46920.0030

Boldface type indicates r < –0.5.

Open in new tab
Table 6.

Correlation Analysis Between Progesterone Levels and Lipids in Patients With a Premature Progesterone Rise and High Level of E2 (E2 ≥3000 pg/mL) at the End of the Follicular Phase

Lipid MoleculerP Value
PC(16:0/18:1)+H−0.35300.0297
SM(d16:0/18:1)+H−0.34990.0313
PC(18:0/18:1)+H−0.40670.0113
PC(20:1/18:2)+H−0.35440.0339
PE(16:0p/18:1)+H−0.44060.0056
LPE(18:0)+H−0.45990.0062
PE(18:0p/18:1)+H−0.49140.0017
Cer(d18:1/16:0)+H−0.39610.0152
TG(16:0/18:2/18:3)+NH40.33990.0368
PE(18:1p/18:1)+H−0.45420.0295
LPC(18:1)+H−0.40010.0128
PI(18:0/18:1)−H−0.42850.0091
PE(16:0p/18:1)−H−0.42750.0074
PS(16:0/19:0)−H−0.65270.0083
PE(16:0/18:1)−H−0.37650.0198
LPE(18:0)−H−0.45410.0042
PI(16:0/18:1)−H−0.48230.0029
LPE(16:0p)−H−0.36740.0386
PE(18:1p/18:1)−H−0.56970.0045
PE(18:0p/18:1)−H−0.46620.0032
PE(18:1/20:3)−H−0.43490.0298
PE(16:0/16:1)−H−0.45580.0169
PI(16:0/22:6)−H−0.43250.0095
LPE(18:1)−H−0.46920.0030
Lipid MoleculerP Value
PC(16:0/18:1)+H−0.35300.0297
SM(d16:0/18:1)+H−0.34990.0313
PC(18:0/18:1)+H−0.40670.0113
PC(20:1/18:2)+H−0.35440.0339
PE(16:0p/18:1)+H−0.44060.0056
LPE(18:0)+H−0.45990.0062
PE(18:0p/18:1)+H−0.49140.0017
Cer(d18:1/16:0)+H−0.39610.0152
TG(16:0/18:2/18:3)+NH40.33990.0368
PE(18:1p/18:1)+H−0.45420.0295
LPC(18:1)+H−0.40010.0128
PI(18:0/18:1)−H−0.42850.0091
PE(16:0p/18:1)−H−0.42750.0074
PS(16:0/19:0)−H−0.65270.0083
PE(16:0/18:1)−H−0.37650.0198
LPE(18:0)−H−0.45410.0042
PI(16:0/18:1)−H−0.48230.0029
LPE(16:0p)−H−0.36740.0386
PE(18:1p/18:1)−H−0.56970.0045
PE(18:0p/18:1)−H−0.46620.0032
PE(18:1/20:3)−H−0.43490.0298
PE(16:0/16:1)−H−0.45580.0169
PI(16:0/22:6)−H−0.43250.0095
LPE(18:1)−H−0.46920.0030

Boldface type indicates r < –0.5.

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Discussion

An elevation in the serum P level during the late follicular phase may still occur without an LH surge in up to 35% and 38% of IVF cycles stimulated with GnRH agonist and antagonist protocols, respectively (19). The elevated peripheral P level in the late follicular phase may lead to asynchrony between the endometrium and the developing embryo (1). However, the underlying mechanism is still unclear. Previous studies have attempted to explain this issue in terms of genomic profiles (11, 20, 21), endometrial morphology, uterine natural killer (NK) cells (22), microRNA analysis, microarray analysis (12), angiogenic factors (23), and epigenetic modifications (24). This study presents the lipidomics profiles of the endometrium between high P and normal P levels on the hCG day by UHPLC-ESI-HRMS. The levels of PC, PE, LPC, LPE, DG, Cer, PI, and PS were significantly decreased in the endometrium during the window of implantation in patients with a premature P rise. Correlation analysis showed that all of the lipids were negatively correlated with high P levels on the hCG day. The correlation analysis between the P levels and lipids showed a stronger negative correlation between PS (16:0/19:0) and PE (18:1p/18:1) according to the correlation coefficient.

The number of days of stimulation, total dose of rFSH, number of oocytes retrieved, and E2 level on the day of hCG administration in the HP group were significantly increased compared with those in the NP group in this study. Previous studies have shown that serum P levels on the day of hCG administration are significantly correlated with the magnitude of the ovarian response to stimulation-associated E2 levels at the time of hCG administration and the number of oocytes retrieved (25, 26). The serum E2 level and number of follicles have predictive value for a premature P rise (27). The possible mechanism of the premature P rise is that FSH promotes P synthesis and output from granulosa cells by promoting the expression and activating enzyme of 3β-hydroxysteroid dehydrogenase (19). In addition, a high E2 level mediates earlier expression of P receptors, leading to the advancement of the endometrium in the late follicular phase (28). On the other hand, there is also negative feedback to downregulate E2 receptors (29).

In our study, the mean estradiol levels on the day of hCG administration were 4886.34 and 6320.12 pg/mL in the NP and HP groups, respectively. Although there was a statistically significant difference between the two groups, the plasma E2 level in the NP group was considered higher. One possible explanation was that the average age of patients in the NP group was lower than that of the study group, although there was no significant difference. The age ranges in the NP and HP groups were 24 to 36 and 27 to 38, respectively. Age is an independent predictor of ovarian reserve and ovarian stimulation outcome (30). Unfortunately, we did not show anti-Müllerian hormone results because some patients did not have their anti-Müllerian hormone levels measured. It is difficult to differentiate the negative effects of E2 and P when they are at supraphysiological levels in stimulated IVF cycles. A previous study showed that estrogen regulated the integrated physiology of cell lipid homeostasis through a specific estrogen receptor (31). An antiestrogenic agent affects the plasma lipid profile (32). To avoid the influence of inconsistent estrogen levels on the metabolic spectrum of the endometrium, the lipid profiles at low levels of E2 (<3000 pg/mL) were investigated. However, we did not find enough samples for statistical analysis of patients with high P levels and low E2 levels on the day of hCG administration. To exclude this confounding factor, we investigated the lipidomics of patients with E2 ≥3000 pg/mL on the hCG day. The lipid profile was similar to the result of the previously unclassified E2 level. Thus, we proposed that P was more detrimental to the endometrium according to these data. On the other hand, the E2 level seems to affect the embryo more negatively than it does the endometrium (33).

In ruminants, P secreted from the corpus luteum induces the accumulation of lipids in endometrial cells. Lipids accumulated in the endometrium are a crucial source of fatty acids for utilization by the conceptus during elongation. Endometrial lipids are exported to the uterine lumen and incorporated by conceptus cells (34). Lipidomics analysis is a rarely used method among in the research on the endometrium (35). A recent study suggested that lipidomic profiling of endometrial fluid may be a valuable tool for identifying the time interval comprising the window of implantation (36). The lipid composition in the endometrium is crucial for embryo implantation. The current study demonstrates that PS (16:0/19:0)−H and PE (18:1p/18:1)−H are closely related to a premature P rise. A previous study showed that the sphingolipid metabolic pathway is important for uterine decidualization. Maternal disturbances in activated sphingolipid metabolism by disruption of sphingosine kinase genes result in a reduction in PE levels and causes uterine hemorrhage and early embryonic lethality (37). Cell activation, injury, and programmed cell apoptosis cause the collapse of PS/PE asymmetry (38). One study showed that antibodies to PE and PS are associated with increased NK cell activity in patients with non-male-factor infertility (39). Several studies have shown abnormal uterine NK cell numbers in patients with poor pregnancy outcomes (40, 41). Peripheral NK cells are related to recurrent spontaneous abortion (42). We hypothesized that lipid changes were closely related to endometrial receptivity. However, studies of NK cells on endometrial receptivity and miscarriage are conflicting. Further experiments are needed to study the specific molecular mechanisms of lipid changes in terms of endometrial receptivity.

In conclusion, alterations in lipid profiles at the window of implantation were studied in patients with a premature P rise at the end of the follicular phase. The levels of lipids including PC, PE, LPC, LPE, DG, Cer, PI, and PS were significantly lower in patients with high progesterone levels on the day of hCG administration. A strong negative correlation was apparent only between PS or PE and progesterone level. The alterations in lipids at the window of implantation suggest an adverse effect of elevated P on endometrial receptivity. Our findings also provide potential targets for endometrial receptivity. Further studies are needed to explore the molecular mechanism of lipid profiling alterations affected by HP levels.

Acknowledgments

Financial Support: This study was financially supported by the National Key Research and Development Program Grant 2017YFE0109900 (to H.B.), the National Natural Science Foundation of China Nos. 81601347 (to J.L.) and 81573489 (to H.B.), the 111 project Grant B16047 (to H.B.), the Natural Science Foundation of Guangdong Province Grant 2017A030311018 (to H.B.), the Key Laboratory Foundation of Guangdong Province Grant 2017B030314030 (to H.B.), and the Guangdong Science and Technology Department No. 2016A020218006 (to J.L.).

Disclosure Summary: The authors have nothing to disclose.

Data Availability: The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.

Abbreviations:

    Abbreviations:
     
  • BMI

    body mass index

    •  
  • Cer

    ceramide

    •  
  • DG

    diacylglycerol

    •  
  • E2

    estradiol

    •  
  • hCG

    human chorionic gonadotropin

    •  
  • HEHP

    samples with high levels of E2 then divided into high progesterone groups

    •  
  • HENP

    samples with high levels of E2 divided into normal progesterone groups

    •  
  • HP

    high progesterone

    •  
  • ICSI

    intracytoplasmic sperm injection

    •  
  • IVF

    in vitro fertilization

    •  
  • LPC

    lysophosphatidylcholine

    •  
  • LPE

    lysophosphatidyl ethanolamine

    •  
  • NK

    natural killer

    •  
  • NP

    normal progesterone

    •  
  • OPLS-DA

    orthogonal projection to latent structures discriminant analysis

    •  
  • P

    progesterone

    •  
  • PC

    phosphatidylcholine

    •  
  • PE

    phosphatidylethanolamine

    •  
  • PI

    phosphatidylinositol

    •  
  • PS

    phosphatidylserine

    •  
  • rFSH

    recombinant FSH

    •  
  • UHPLC-ESI-HRMS

    ultra-high-performance liquid chromatography with high-resolution mass spectrometry

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Author notes

J.L. and Y.G. contributed equally to this work.

Copyright © 2019 Endocrine Society
Issue Section:
Reproductive Biology and Sex-Based Medicine
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