LC-MS detected universal metabolites of human day 3 embryos with distinct implantation outcomes for Cook Medical and Vitrolife culture medium

doi:10.21203/rs.3.rs-2339954/v1

Background

To implore the universal metabolites from metabolomic profiling of spent embryo culture medium (SECM) correlated to day 3 embryo’s implantation potential in two commercial culture medium (Cook Medical and Vitrolife).

Methods

This investigation was a prospective randomized study. Patients undergoing IVF-ET and having utilizable day 3 embryos were recruited. Patients were randomized for embryo culture in Cook Medical or Vitrolife medium. On day 1 each zygote of patients was individually cultured in a 30 µL microdroplet until day 3. Then SECM from each microdroplet was collected within 2 hours after the embryo was utilized (transferred or frozen) and detected by Liquid Chromatography-Mass Spectrometry (LC-MS).

Results

For Vitrolife medium, 3 batches of total 69 SECM samples were collected. According to the embryo’s implantation outcome, 17 were successfully implanted group, while 52 were failed implanted group. 147 metabolomic irons presenting significantly different level between the two groups were identified as the Vitrolife metabolomic profiling set. For Cook Medical medium, 2 batches of total 37 SECM samples were collected. According to the embryo’s implantation outcome, 14 were successfully implanted group, while 23 were failed implanted group. 435 metabolomic irons presenting significantly different level between the two groups as the Cook Medical metabolomic profiling set. There were 66 universal metabolites between the Vitrolife and Cook Medical metabolomic profiling set and the ion with m/z = 121.029 was the most reproduciable signature, which was identified to be benzoic acid.

Conclusions

Metabolomic profiling in SECM correlated to day 3 embryo’s implantation potential in Vitrolife and Cook Medical medium could be sensitively detected by LC-MS approach. The universal metabolites for the two commercial medium, such as benzoic acid, may provide a potential solid adjunct to day 3 embryo’s evaluation.

LC-MS

metabolomic profiling

embryo spent culture medium

implantaion

Cook Medical and Vitrolife culture medium (CM) are the most two commonly used commercial medium in in vitro fertilization - embryo transfer (IVF-ET) laboratory for their ease of acquisition. The developmental outcome of human in vitro embryos in these two CM has been compared in several studies with contradictory results[1–3], which makes it difficult to get a consens on which medium we should use. The composition of Cook Medical and Vitrolife CM varies considerably such as in pyruvate, lactate and amino acids [4]. The concentration of amino acids in Cook Medical cleavage medium was much higher than that in Vitrolife cleavage medium, while the ratio of lactate to pyruvate (L:P) in G1 (Vitrolife Cleavage medium) was about 7-fold higher (36 vs 5) than that in Cook cleavage medium [4]. Since both the different amino acid profiling and the interconversion between lactate and pyruvate can affect the embryo metabolomic behavior [5], the metabolomic profiling of spent embryo culture medium (SECM) relevant with embryo implantation potential varied according to corresponding culture medium. So the universal metabolites relevant with embryo implantation potential between Cook Medical and Vitrolife will become more reasonable biomarkers for embryo viability. However, to the best of our knowledge, it is still poorly studied. To find universal metabolites relevant with embryo implantation potential between Cook Medical and Vitrolife, the SECM samples of embryos individually cultivated from zygotes to day 3 using Cook Medical and Vitrolife CM were firstly collected separately, then metabolomic profiling were tested by LC-MS for these two medium to implore the metabolites in SECM relavent with day 3 embryo’s implantation outcome, finally, the similarities between these two medium’s remarkable metabolites were compared and identified.

2.1 Patients selection and study design

A prospective radomized study was designed from March 2019 to March 2020. Patients who intended to undergo IVF-ET and day 3 embryo transfer (including fresh and thawing embryo transfer cycle) were included. Exclusion criteria were as follows: patients who got none cultivated embryos (none oocyte or only had degenerated oocytes after oocytes retrieval; none normally fertilized zygotes (with two pronuclei 16–18h after insemination)); patients diagnosed endometrial dysplasia before transfer (endometrial thickness less than 6 mm or no typical "trilinear sign" at the implantation window); and patients with singleton pregnancy after double embryos transfer. This study was approved by the Ethics Review Board of the Peking University People’s Hospital (2018PHB061-01). Each participant was informed in detail about the study’s procedures and risks, and the consent was obtained.

2.2 Ovarian stimulation and oocyte retrieval

Ovarian stimulation was performed on each participant under different controlled ovarian stimulation (COS) regimens (i.e., the long, antagonist, and others (minimal-stimulation, natural, luteal phase stimulation, and progestin-primed stimulation)) according to their characteristics and responses during previous IVF cycles or ovulation induction. Follicular maturation was monitored by ultrasound examination and ovulation was induced with 5000–10,000 IU of human chorionic gonadotropin (hCG; Choragon, Ferring, Switzerland), alone or in combination with 0.2 mg triptorelin acetate (Ferring) if there was at least two follicles of ≥ 18 mm in diameter. Oocytes were retrieved by ultrasonography-guided transvaginal aspiration of the follicles at 36h after hCG administration.

2.3 Insemination, embryo cultivation and processing

At 40h after hCG injection, the retrieved oocytes were inseminated by conventional IVF or single sperm intracytoplasmic injection (ICSI) according to clinician’ instruction. In brief, conventional IVF was performed by transferring a proper amount of prepared sperm into the dish where the oocytes were incubated. For ICSI, the oocytes’cumulus cells were removed at 1h after retrieval by gently pipetting, following a short, enzymatic digestion in hyaluronidase (Sage). The denuded oocytes were incubated in the cleavage medium and were then subjected to sperm injection in a gamete manipulation buffer; the oocytes were immediately transferred to the culture dish in cleavage medium post-ICSI.

At 16-18h after insemination, the number of pronuclei in zygotes were observed under the microscope and each normally fertilized zygote (two pronuclei) was transferred into a new microdroplet of 30 µL cleavage medium (Cook Medical or Vitrolife) individually for culture until day 3. Then embryos were evaluated morphologically under the inverted microscope and embryos scored grade I or II by the modified Pruissant scoring criteria [6]were transferred or frozen. Frozen embryos were then thawed and transferred in patients’ thawing cycle after oocytes retrieval. 14 days after embryo transferring, hCG in blood was tested to determine whether the patient was clinical pregnancy or not, and ultrasound was performed on 28 days to determine the number of fetal sacs.

2.4 SECM Samples collecting and grouping

Within 2 hours after each day 3 embryo was utilized (transferred or frozen), 25 µL of corresponding SECM was removed from the microdroplet, put into a small brown glass vial, marked well and stored in the refrigerator at -80 degrees. The SECM samples were grouped according to the follow-up results: if the patient was successfully pregnant after transfer and the number of fetal sacs visible under ultrasound was the same as transferred embryos, their transferred day 3 embryos were successfully implanted and the corresponding SECM samples were classified as the successful implantation group (s-implanting group); if the patient was not successfully pregnant, their transferred day 3 embryos failed and the corresponding SECM samples were classified as the failed implantation group (f-implanting group).

2.5 Samples preparation and metabolomic analysis by LC-MS

Metabolites were extracted from SECM samples with cold methanol. 180 uL of cold methanol was added into 20 uL of SECM, and insoluble materials including human serum albumin were centrifuged at 14,000 × g for 15 min, and 180 ul of resulting supernatants were dried under vacumm. Samples were resuspended in 36 µl of 1:1 methanol/water solution (v/v) for liquid chromatography-high resolution mass spectrometry.

The LC-MS analysis was performed on a Dionex Ultimate 3000 UHPLC (Thermo, USA) coupled with a Q-Exactive mass spectrometer (Thermo, USA). Compounds were separated on an XBridge Amide column (100 × 4.6 mm, 3.5 µm, Waters, Milford, MA, USA) with the following gradient: 0–5 min, 85–42% B; 5–16 min, 42–5% B; 16–24 min, 5% B; 24–25 min, 5–85% B; 25–32 min, 85% B. Buffer A comprised 20 mM ammonium acetate (pH 9.4) in water, and buffer B was acetonitrile (LC-MS grade). The mass resolution was 70,000, automatic gain control (AGC) target was 1×10⁶, and maximum inject time was 100 ms. The flow rates of sheath gas, aux gas and sweep gas were 35, 15 and 2 units, respectively. Negative mode and full MS scan type were chosen for metabolomic analysis.

2.6 Data processing

Raw data of metabolomic analysis was performed by pairwise mode on XCMSonline (https://xcmsonline.scripps.edu/). Maximal tolerated m/z deviation in consecutive scans was set as 5 ppm, and tolerance for database search was set as 15 ppm. Signal/Noise threshold was 4. Welch t-test was used for statistical test with p-value threshold of 0.05 and peak area fold change threshold of 1.5. Remove the software misidentified differential metabolites based on peak shape. Metabolite structure and function information was searched in the database HMDB (www.hmdb.ca) and KEGG (https://www.kegg.jp).

2.7 Structural analysis of characteristic metabolites

For SECM characteristic metabolite structural analysis, MS/MS fragmentation was performed using HCD collision mode by select top 20 most intense ions with the resolution of 17500. The isolation window was 4.0 m/z, charge was 1-, collision energy was NCE 40, dynamic exclusion was 10 s. The retention times and MS/MS fragments were matched based on standard compounds.

2.8 Statistical analysis

Clinical variables in Cook Medical and Vitrolife culture medium were separately analyzed by the same statitical methods. In brief, continuous variables were presented as mean ± standard and were analyzed by unpaired t-test between the s-implanting and f-implanting group. Categorical variables were expressed in frequency and percentage and were analyzed by Chi square test between the two groups. P values less than 0.05 (two-sided) were considered statistically significant.

3.1 An efficient LC-MS approach for distinguishing Cook Medical and Vitrolife SECM for day 3 embryos.

Fresh culture medium without embryo culture and SECM samples were used to establish the procedures of sample collecting, preparing and detection by LC-MS approach. Firstly, metabolomic sample preparing and LC-MS method was established using fresh Cook Medical and Vitrolife culture medium without embryo culture. LC separated the metabolites extracted from the fresh culture medium effectively. With the excellent qualitative and quantitative ability of high-resolution mass spectrometry, the culture medium components can be identified, including high content of non-essential amino acids and nutrient molecules (Fig. 1).

Methanol was used as protein precipitation reagent, and the ratio was optimized at 3:1, 4:1 and 9:1 (methanol: medium sample, v/v). MS signal of several amino acids were extracted and method precision was evaluated by calculate the relative standard deviations. Among them, the MS signal of 9:1 treatment was the best, and the relative standard deviations (RSDs) of the mentioned amino acids were within 5% (Supplemental Table 1).

Next, 10 SECM samples were used to evaluated the impact of SECM collecting. There was no metabolomic difference between 2 SECM samples from a same microdroplet collected by 2 different embryologists under the same protocol (Fig. 2).

Metabolite profiling was performed on the medium before and after embryo culture, and it was found that the content of pyruvate and lactate in the medium after embryo culture decreased (Fig. 3), which indicated LC-MS could quantitativly revealed the metabolites’ content in SECM.

After the above procedures about sample collecting, preparing and detection by LC-MS were tested, the approach was established, 20 SECM samples from either Cook Medical or Vitrolife medium were collected and detected by LC-MS. Interactive principal components analysis (iPCA) showed a clear clustering of SECM samples from Cook and Vitrolife medium (Fig. 4), which indicated LC-MS could efficiently distinguished SECM samples from these two commercial medium.

3.2 Baseline Characteristics of Patients and Embryos for Vitrolife medium and Cook Medical medium.

For Vitrolife medium, a total of 52 patients were enrolled, and their 69 embryos were transfered (17 patients were transfered 2 embryos; and 35 patients were transfered with one single embryo) in the study. The spent culture medium of the 69 embryos were collected successfully and analyzed by LC-MS. 12 patients (17 embryos) became pregnant successfully after embryo transfer (s-implanting group), while the other 40 patients (52 embryos) failed (f-implanting group) (Fig. 5). The demographic and clinical characteristics of the two groups are listed in Table 1. There were no significant differences between the two groups in mean age, body mass index (BMI) of the female patients, the level of AMH, ovary stimulation duration, number of antral follicles and number of retrieved oocytes. The mean age of female’s parterner from f-implanting group was significantly older than that from s-implanting group (38.90 vs 35, P = 0.021). The infertility duration of f-implanting group was significantly longer than that from s-implanting group (3.68 vs 1.88, P = 0.003). The thickness of the patients’ endometrium before transferring from s-implanting group was significantly thicker than that from f-implanting group (11.13vs 8.86, P = 0.003. 66.7% and 42.5% patients in s-implanting group and f-implanting group were diagnosed primary infertility without significant difference. Approximately 94.1%, and 94.2% embryos transferred were 7–10 blastomeres in s-implanting and f-implanting group. Approximately 70.6%, and 53.8% transferred embryos in s-implanting and f-implanting group were morphologically evaluated as grade I. The proportion of 7–10 blastomeres embryos and morphological grade I embryos had no significant differences among the two groups.

For Cook Medical medium, a total of 26 patients were enrolled, and their 37 embryos were transfered (11 patients were transfered 2 embryos; and 15 patients were transfered with one single embryo) in the study. The spent culture medium of the 37 embryos were collected successfully and analyzed by LC-MS. 10 patients (14 embryos) became pregnant successfully after embryo transfer (s-implanting group), while the other 16 patients (23 embryos) failed (f-implanting group) (Fig. 5). The demographic and clinical characteristics of the two groups are listed in Table 1. There were no significant differences between the two groups in mean ages of the female and their husbands, female patients’body mass index (BMI), infertility duration, the level of AMH, ovary stimulation duration, number of antral follicles and number of retrieved oocytes and the mean thickness of the patients’ endometrium before transferring. 50% and 46.7% patients in s-implanting group and f-implanting group were diagnosed primary infertility without significant difference. Approximately 100%, and 95.7% embryos transferred were 7–10 blastomeres in s-implanting and f-implanting group. Approximately 50% and 39.1% transferred embryos in s-implanting and f-implanting group were morphologically evaluated as grade I. The proportion of 7–10 blastomeres embryos and morphological grade I embryos had no significant differences among the two groups.

Table 1

Demographic and clinical characteristics of the patients undergoing day 3 embryo transfer and spent embryo culture medium collecting for Vitrolife and Cook Medical
	Vitrolife			Cook Medical
	s-implanting (n = 12)	f-implanting (n = 40)	P value	s-implanting (n = 10)	f-implanting (n = 16)		P value
Maternal age (years)	34.25 ± 3.62	36.50 ± 5.28	0.174	33.60 ± 2.50	32.50 ± 4.93		0.521
Paternal age (years)	35.00 ± 4.13	38.90 ± 6.76	0.021	33.70 ± 2.87		34.81 ± 5.91	0.586
Maternal BMI (kg/m2) a	22.32 ± 2.18	22.68 ± 3.67	0.761	23.62 ± 3.61		26.14 ± 4.72	0.166
Infertility duration (years)	1.88 ± 1.21	3.68 ± 2.88	0.003	4.00 ± 2.67		3.46 ± 2.68	0.619
AMH (ng/ml)	2.73 ± 2.14	2.24 ± 1.99	0.498	2.76 ± 1.86		2.88 ± 3.41	0.922
Ovary stimulation duration (days)	9.25 ± 2.42	8.85 ± 1.98	0.563	9.60 ± 0.97		9.75 ± 1.61	0.793
Antral follicles (numbers)	10.00 ± 7.20	7.55 ± 5.26	0.201	10.10 ± 4.33		10.38 ± 8.75	0.927
Retrieved oocytes (numbers)	8.58 ± 6.49	5.38 ± 3.66	0.125	7.40 ± 3.92		6.63 ± 3.67	0.614
Endometrial thickness b (mm)	11.13 ± 2.30	8.86 ± 2.16	0.003	10.13 ± 2.34		9.42 ± 1.86	0.403
Primary infertility, n (%)	8(66.7)	17(42.5)	0.142	5 (50)		7 (46.7)	0.870
No. of embryos transferred on day 3	s-implanting (n = 17)	f-implanting (n = 52)		s-implanting (n = 14)		f-implanting (n = 23)
7–10 cell embryo rate, n (%) c	16(94.1)	49(94.2)	0.986	14 (100)		22 (95.7)	0.429
Morphological grade I embryo rate, n (%) d	12(70.6 )	28(53.8)	0.225	7 (50)		9 (39.1)	0.517

a BMI is the abbreviation of body mass index;

b Endometrial thickness: the thickness of the endometrium on the LH surge day.

C Proportion of embryos with 7–10 blastomeres on day 3

d Proportion of morphological grade I embryos

Italic P-value indicates significantly different

3.3 Metabolomic ions with different level between s-implanting and f-implanting group were detected in Vitrolife and Cook Medical SECM.

The 69 and 37 SECM samples were collected and analyzed by LC-MS within 3 and 2 batches respectively from Vitrolife and Cook Medical medium and a series of metabolomic ions with different level (a fold change of ≥ 1.5 and p < 0.05 by univariate statistics) were found (Supplemental Fig. 5). Among the differential metabolites in 2 batches of Cook Medical medium and 3 batches of Vitrolife medium, 66 metabolites overlapped at least in 2 batches (Fig. 6). The ion with m/z 121.029 was the only universal metabilite which had the similar trend between batches (Fig. 7) and was identified as benzoic acid by structure analysis (Fig. 8A, B) .

A reliable noninvasive method to choose the best candidate with real implantation potential for transfer from two embryos with equal morphological qualities is still lacking clinically, which hampered the improvement of single embryo transfer stratege. Spent embryo culture medium could be collected noninvasively after embryo was cultivated, and its metabolomic profiling relevent with embryo implantation potential could be detected by a range of spectroscopic methods including Fourier Transform infrared spectroscopy (FTIR), near-infrared spectroscopy (NIR), Raman spectroscopy [7, 8], NMR [9] and liquid chromatography Mass-spectrometry (LC-MS) [10, 11]. Then an approach basing on one of these metabolomic profiling detection methods provids a potential adjunct to embryo morphological assessment. However, NIR spectroscopy detection of spent embryo culture media for day 2,3 and 5 embryo had been reported to be useless in adjunct to morphology evaluation [12, 13]. Detection sensitivity of each metabolomic profiling method was different and it may affect its efficiency of predicting embryo viability [14]. Therefore, high sensitive metabolomic profiling detection method such as LC-MS may provide more information. In 2022, Eldarov C et al performed HPLC-MS on SECM samples from day 3 embryos of the first morphological class with corresponding transfer outcomes and they selected 50 molecular irons with a significant fold change between the s-implanting and f-implanting group [11]. They concluded HPLC-MS for SECM metabolomic analysis could be a powerful and sensitive tool for embryo implantation potential. In our study, we found 147 and 435 molecular irons (Supplemental Table 2) correlate with day 3 embryo implantation outcome for Vitrolife and COOK Medical medium respectively using LC-MS, which also indicated the robustness of LC-MS on SECM detection.

Nowadays since there has been insufficient evidence to support or refute the use of any specific commercial culture medium, two or more commercial medium were simutaneously used in several IVF labaratories. Cook Medical and Vitrolife were the two most common used medium type for human embryo cultivation in Chinese IVF labaratories, including our labaratory. This provided us the opportunity to evaluate the difference between metabolomic profiling according to Cook Medical and Vitrolife culture medium under the similar other culture condition and metabolomic profiling detection method. 66 universal metabolomic sets between SECM from Cook Medical and Vitrolife culture medium were found in our study (Supplemental Table 3). However, none of these 66 metabolomic sets exsited in the metabolomic sets related with day 3 implantation potential from Eldarov’s study. Except the different types of commercial medium used between our and their study, other culture conditions such as incubator also may contribute to lack of consistency, and this also indicated that it was difficult to find universal metabolomic ions between studies with different culture condition and metabilomic profiling detection methods.

In the 66 metabolomic sets found in our study, the metabolomic ion with m/z 121.029 was the highest universal molecule. Not only between batches but also between Cook Medical and Vitrolife culture medium, the level of m/z 121.029 in SECM from s-implanting group was lower than that from f-implanting group. The metabolomic ion with m/z 121.029 was identified as benzoic acid by structure analysis (Fig. 8A, B). To the best of our knowledge, it is the first time reported that the concentration of benzoic acid in spent embryo culture medium correlated with day 3 embryo implantation potential and was an universal metabolomic biomarker between two commercial medium. As reported before, though the composition of Cook Medical and Vitrolife cleavage medium varied on the concentration of amino acids, phenylalanine was the commom supplement for these two medium, and we indeeded detected phenylalanine both in Cook Medical and Vitrolife cleavage culture medium before embryo cultivation (Fig. 8C, D). Also it was interesting that we did not detect benzoic acid neither in Cook Medical nor Vitrolife cleavage culture medium before embryo cultivation (Fig. 8A). So We speculate that from zygote to day 3, embryo could biosynthesized benzoic acid to its microdroplet circumstance from phenylalanine via CoA-dependent-β-oxidation pathway like plants [15][16]. In this pathway reactive oxidative stess (ROS) were also produced and finally impaired the day 3 embryo’s developmental potential. So we found a strong negtive association between the day 3 embryo’s implantation potential and the concentration of benzioc acid in its SECM. This also indicated that phenylalanine in culture medium may be harmful to the first three day’s embryonic development, which is consistent with Bavister’s study. They founded phenylalanine reduced mean cell number of one-cell hamster embryos’ development when added to basal culture medium [17].

To the best of our knowledge, the present study revealed for the first time the concentration of benzoic acid in spent embryo culture medium correlated negatively with day 3 embryo implantation potential and indicated phenylalanine in the culture medium could be the origin, which provides information of improving the formulation of human embryo culture medium. However, there were limitations for this study. Firstly, the sample size of SECM was relatively limited. One of the reasons was that with improvement of blastocysts culture technology, the proportion of day 3 embryo transfer cycle gradually declined in our center. Secondly, The size between the s-implanting and f-implanting group was not equal, which may affect our result. The low efficiency of single day 3 embryo transfer contributed to this. Furthermore, the level of benzoic acid are needed to be verified in large size samples in future and its related biosynthesized pathway in human embryos should be studied furtherly.

Metabolomic profiling in SECM correlated to day 3 embryo’s implantation potential in Vitrolife and Cook Medical medium could be sensitively detected by LC-MS approach. The universal metabolites for the two commercial medium, such as benzoic acid, may provide a potential solid adjunct to day 3 embryo’s evaluation.

SECM: spent embryo culture medium; CM: culture medium; IVF-ET: In vitro fertilization embryo transfer; COS: controlled ovarian stimulation; LC-MS: Liquid Chromatography-Mass Spectrometry; hCG: human chorionic gonadotropin; ICSI: intracytoplasmic sperm injection; AGC: automatic gain control; RSDs: relative standard deviations; iPCA: Interactive principal components analysis;FTIR: Fourier Transform infrared spectroscopy; NIR: near-infrared spectroscopy; ROS: reactive oxidative stess.

Ethics approval and consent to participate

This study was approved by the Ethics Review Board of the Peking University People’s Hospital (2018PHB061-01). Each participant was informed in detail about the study’s procedures and risks, and the consent was obtained. The research was conducted in accordance with the World Medical Association's Declaration of Helsinki.

Consent for publication

All participants provided written informed consent for publication of this study.

Availability of data and materials

The datasets used or analyzed in this study are available from the corresponding authors on reasonable request.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

This work was supported by Beijing Natural Science Foundation (7222200 and 7222197) and the Chinese Medical Association Special funding Project for Clinical Medical Scientific Research (17020530722).

Authors' contributions

SC, CX and SH proposed the concept and designed the study. WP, LR and DSN contributed to the acquisition of data. FM supervised data collection. LY and CYN performed the LC-MS experiments. SC and LY wrote the initial manuscript, CX, ZYL and SH revised the manuscript. All authors provided input for preparation of the manuscript.

Acknowledgments

The authors thank AiMi Academic Services (www.aimieditor.com) for English language editing and review services.

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No competing interests reported.

LC-MS detected universal metabolites of human day 3 embryos with distinct implantation outcomes for Cook Medical and Vitrolife culture medium

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

1. Background

2. Materials and Methods

2.1 Patients selection and study design

2.2 Ovarian stimulation and oocyte retrieval

2.3 Insemination, embryo cultivation and processing

2.4 SECM Samples collecting and grouping

2.5 Samples preparation and metabolomic analysis by LC-MS

2.6 Data processing

2.7 Structural analysis of characteristic metabolites

2.8 Statistical analysis

3. Results

3.1 An efficient LC-MS approach for distinguishing Cook Medical and Vitrolife SECM for day 3 embryos.

3.2 Baseline Characteristics of Patients and Embryos for Vitrolife medium and Cook Medical medium.

3.3 Metabolomic ions with different level between s-implanting and f-implanting group were detected in Vitrolife and Cook Medical SECM.

4. Discussion

5. Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1