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ABSTRACT There exist some patients who face recurrent total fertilization failure during assisted reproduction treatment, but the pathological mechanism underlying is elusive. Here, by using

sc-RNA-seq method, the transcriptome profiles of ten abnormally fertilized zygotes were assessed, including five zygotes from one patient with recurrent Poly-PN zygotes, and five zygotes

from a patient with pronuclear fusion failure. Four zygotes with three pronuclear (Tri-PN) were collected from four different patients as controls. After that, we identified 951 and 1697

significantly differentially expressed genes (SDEGs) in Poly-PN and PN arrest zygotes, respectively as compared with the control group. KEGG analyses indicated down regulated genes in the

Poly-PN group included oocyte meiosis related genes, such as _PPP2R1B, YWHAZ, MAD2L1, SPDYC, SKP1_ and _CDC27_, together with genes associated with RNA processing, such as _SF3B1, LOC645691,

MAGOHB, PHF5A, PRPF18, DDX5, THOC1_ and _BAT1_. In contrast, down regulated genes in the PN arrest group, included cell cycle genes, such as _E2F4, DBF4, YWHAB, SKP2, CDC23, SMC3, CDC25A,

CCND3, BUB1B, MDM2, CCNA2_ and _CDC7_, together with homologous recombination related genes, such as _NBN, XRCC3, SHFM1, RAD54B_ and _RAD51_. Thus, our work provides a better understanding

of transcriptome profiles underlying RTFF, although it based on a limited number of patients. SIMILAR CONTENT BEING VIEWED BY OTHERS _ARHGAP26_ DEFICIENCY DRIVES THE OOCYTE ANEUPLOIDY AND

EARLY EMBRYONIC DEVELOPMENT FAILURE Article 23 September 2024 SINGLE CELL RNA-SEQ REVEALS GENES VITAL TO IN VITRO FERTILIZED EMBRYOS AND PARTHENOTES IN PIGS Article Open access 13 July 2021

AN INTEGRATED ANALYSIS OF MULTIPLE DATASETS REVEALS NOVEL GENE SIGNATURES IN HUMAN GRANULOSA CELLS Article Open access 06 September 2024 INTRODUCTION Despite nearly forty years of scientific

and clinical advance in the field of assisted reproduction, there still exist some rarely patients, even though rarely occur, who have to face recurrent total fertilization failure (RTFF)

without any visual precautionary indicator1,2,3, even some of them could be rescued by assisted oocyte activation4. Therefore, elucidating the internal mechanism of fertilization failure is

of great importance for these patients. However, until now, the relevant etiological analysis was often restricted to morphology during IVF, such as immature oocytes5, improper meiosis6,

zygotes with abnormal pronuclei7, and di-pronuclear zygote failing mitotic cleavage8. Due to small amount of material available, deciphering mechanisms underlying these defects remain

technical challenging. Recent technical advances in single-cell sequencing open a new era for exploring the biological state of a single cell at both the DNA and RNA levels for studying

variations in genome9,10, transcriptome, and epigenome11 separately or in parallel12. Originally adopted by Surani’s research team13, this approach has been applied successfully in

discriminating cell types14,15,16,17,18,19, elucidating regulatory circuits20, and investigating tumor heterogeneity21,22. In reproductive biology fields, this technique has been used for

screening transcriptome of tissues23 and germline cells at different stages24,25,26,27,28,29. The single cell sequencing technique has great potential in clinical implication30,31,32,

especially in the diagnosis for clarifying the molecular mechanisms of fertilization failure at a single cell resolution. So the aim of this work was to characterize the pathological changes

of human zygotes with RTFF at the transcriptional level. RESULTS CLINICAL TREATMENT HISTORY OF THE RTFF PATIENTS As clinical treatment history shown (Table 1), one patient experienced two

stimulated cycles under different procedures with 4 and 5 Poly-PN fertilized eggs after ICSI treatment, respectively. Another patient had all zygotes with PN arrest, with 18, 7 and 9 matured

oocytes retrieved separately in three cycles although three different ovarian stimulation procedures were employed each time. Moreover, There were no significant differences in serum levels

of FSH, LH, E2 and progesterone at baseline and trigger day in patients with Poly-PN, PN arrest, and the control groups (Supplementary Fig. 1), indicating that the observed defects in the

zygotes were more likely associated with oocyte original molecular defects rather than ovarian stimulation protocol. TRANSCRIPTOME PROFILES IN POLY-PN AND PN-ARREST ZYGOTES It is crucial for

oocyte to accumulate indispensable mRNAs to ensure its later use for fertilization and subsequent cell division before the zygotic gene activation33. As the scarce of the oocytes for RTFF

patients, it was difficult to collect enough donated oocytes for our study. Therefore, we investigate the transcriptome profile using the unfertilized oocyte after clinical treatment. The

procedure of our work was shown in Fig. 1. After sequencing using Illumina HiSeq 2,500 sequencer, we obtain about 142 million clean reads, of which 116 million clean reads mapped to human

genome reference. On average, 15,058, 14,995 and 17,713 genes (FPKM ≥ 0.1), 10,471, 10,451 and 10,289 genes (FPKM ≥ 1) or 4,539, 4,410 and 3,630 genes (FPKM ≥ 10) were acquired in Tri-PN,

Poly-PN and PN arrest groups, respectively (Fig. 2a). These results were consistent with the data from a previous study24, implying that our technology has the similar sensitivity and

coverage. To compare the global transcriptome profiles of unfertilized eggs or zygotes among different groups, we analyzed data by hierarchical clustering, and the results indicated that 14

zygotes from 3 groups were clustered into corresponding groups and separated from each other (Fig. 2b). Four Tri-PN zygotes from different patients have the similar transcriptome profile in

spite of the heterogeneity in patient source. Interestingly, we also found that five PN arrest zygotes clustered closer with four Tri-PN zygotes, but away from the five Poly-PN zygotes. This

finding indicated that the underlying mechanisms was quite different between the Poly-PN and PN arrest group. To clarify underlying mechanisms, we analyzed major differences of expression

profile among different groups. To rule out technical errors causing artifacts of gene expression, all reference genes with average FPKM > 0.5 in any of three groups were used for

subsequent analysis. According to the criteria of fold change > 2 or < 0.5 and _P_ < 0.001, 951 (227 up regulated and 724 down regulated) and 1,697 genes (205 up regulated and 1,492

down regulated) were found to be significantly differentially expressed genes (SDEGs) in Poly-PN and PN arrest zygotes, respectively, as compared with the control group (Fig. 2c,d and

Supplementary Tables 1 and 2). THE SDEGS ARE INVOLVED IN DIFFERENT BIOLOGICAL PROCESSES BETWEEN POLY-PN AND PN ARREST ZYGOTES In order to clarify the biological function of these

differential expressed genes, the KEGG analyses were applied to SDEGs of Poly-PN and PN arrest zygotes, separately. As the results shown (Table 2), for the up regulated genes, there was a

enrichment of genes whose products are related to RNA processing and translation, such as RNA splicing (_P_-value = 1.90 × 10−2 for Poly-PN) or Ribosome biogenesis (_P_-value = 5.80 × 10−9

for Poly-PN and 1.10 × 10−84 for PN arrest), together with energy consuming related items, such as Huntington’s disease (_P_-value = 6.40 × 10−3 for Poly-PN), Parkinson’s disease (_P_-value 

= 1.20 × 10−3 for PN arrest) and Oxidative phosphorylation (_P_-value = 5.20 × 10−5 for PN arrest). Genes involved in Wnt signaling pathway (_P_-value = 1.00 × 10−2 for Poly-PN), Notch

signaling pathway (_P_-value = 2.00 × 10−2 for Poly-PN) and some other signaling pathways in cancer were also enriched (Table 2 and Supplementary Tables 3 and 4). For down regulated genes,

the Poly-PN specific down regulated genes were mainly enriched in Oocyte meiosis (_P_-value = 3.60 × 10−2), Spliceosome (_P_-value = 4.00 × 10−3), Pyrimidine metabolism (_P_-value = 1.60 × 

10−2), Citrate cycle (_P-_value = 3.80 × 10−3) (Table 3 and Supplementary Table 5). Whereas, the PN arrest specific down regulated genes mainly belonged to Cell cycle (_P_-value = 7.60 × 

10−3), Homologous recombination (_P_-value = 2.40 × 10−2) and Amino sugar or nucleotide sugar metabolism (_P_-value = 4.60 × 10−2) (Table 3 and Supplementary Table 6). Furthermore, The SDEGs

down regulated overlapped in both of this two groups were mainly related to Basal transcription factors (_P_-value = 3.10 × 10−2 for Poly-PN and 4.20 × 10−3 for PN arrest), Ubiquitin

mediated proteolysis (_P_-value = 4.70 × 10−2 for Poly-PN and 7.70 × 10−3 for PN arrest) and Glycan biosynthesis (_P_-value = 2.00 × 10−2 for Poly-PN and 3.10 × 10−2 for PN arrest). Among

the total 1,956 down regulated genes in different annotations, there were about 464 (23.7%) genes specifically down regulated in Poly PN, 1,233 (63.0%) genes specifically down regulated in

PN arrest and only 259 (13.3%) genes down regulated overlap in both of this two groups (Fig. 3a). The Poly-PN specific down regulated genes included oocyte meiosis related genes such as

Protein Phosphatase (_PPP2R1B_), _YWHAZ, MAD2L1, SPDYC, SKP1_ and _CDC27_ (Fig. 3b). Certain genes associated with RNA processing, such as those encoding splicing factor genes _SF3B1,

LOC645691, MAGOHB, PHF5A, PRPF18, DDX5, THOC1_ and _BAT1_ were also down regulated (Fig. 3c), perhaps contributing to the fertilization failure in Poly-PN group. In contrast, the PN arrest

specific down regulated genes, such as _E2F4_, _DBF4, YWHAB, SKP2, CDC23, SMC3, CDC25A, CCND3, BUB1B, MDM2, CCNA2, CDC7_ were involved in Cell cycle (Fig. 3d) and _NBN, XRCC3, SHFM1, RAD54B,

RAD51_ were Homologous recombination related genes (Fig. 3e). These results implied that the Poly-PN might have defects during oocyte meiosis, whereas defects of PN arrest zygotes were

involved in Cell cycle and Homologous recombination. FUNCTIONAL VALIDATION OF THE SELECTED GENES BY GENE KNOCK DOWN We randomly chose two of these meiosis related genes (PPP2CA and SKP1) and

validated their function in mice oocyte, the results indicated that both PPP2CA and SKP1 knock down did not show any difference with the corresponding control group in either oocyte

maturation or fertilization (Fig. 4a,b). In order to clarify the mechanism underlying, we analyzed available published single cell RNA-Seq data sets corresponding to fertilization process of

human and mice, focusing on the 43 selected genes, which enriched in Meiosis, Spliceosome, Cell cycle and Homologous recombination items separately. As the results shown (Fig. 4c), human

and mice have very different expression patterns of the selected genes during their fertilization process. Thus, more clinical cases, but not the mice, might be an ideal model for validation

of the function of these selected genes in future. DISCUSSION Some patients have oocytes incapable of completing the whole process of fertilization, including defective sperm entry, oocyte

activation, pronuclear formation or fusion34, as well as some failure in mitotic division35. In this work, we profiled the transcriptome of the Poly-PN and PN arrest zygotes from two

patients with RTFF, and found Poly-PN zygotes showed defects in Meiosis and RNA processing and PN arrest zygotes had defects in Cell cycle and DNA homologous recombination. For meiosis,

oocytes need to undergo meiotic DNA replication and homologous chromosomes segregation, and then arrest in metaphase of meiosis II awaiting fertilization33. After sperm penetration, oocyte

resumes meiosis and segregates sister chromatids and completes the meiosis II. So the differentially expressed genes in Poly-PN might play critical roles in this biological process. For

example, the subunit of the SCF E3 ubiquitin ligase (SKP1) has been reported to be important in the progression of recombination during oocyte meiosis36. The APC core subunit (CDC27) and the

checkpoint protein (MAD2) play critical roles in segregating sister chromatids during oocyte meiosis37. Similarly, some other genes down regulated in Poly-PN zygotes, such as Protein

Phosphatase (_PPP2R1B_), _YWHAZ_ and _SPDYC_ were also in associated with meiosis38,39. Furthermore, during oocyte maturation, it also needs to accumulate sufficient maternal RNA to ensure

oocyte maturation, fertilization and subsequently embryo development until the embryonic genome is activated40. So certain RNA processing genes identified in Poly-PN zygotes, such as those

encoding splicing factor genes _THOC1_41, _SF3B1_42, _LOC645691_, exon junction complex core component related gene (_MAGOHB_)43, PHD finger-like domain-containing protein 5 A (_PHF5A_)44,

some pre-mRNA processing factor 18 related gene (_PRPF18_)45 and RNA helicases related genes (_DDX5_ and _BAT1_)46,47, are involved in regulating RNA secondary structure and pre-mRNA

splicing, which might be responsible for RNA maturation during oocyte meiosis. Upon fertilization, the zygotes undergoes chromatin remodeling, genomes reprogramming or DNA repairing, and the

cell cycle machinery must be switched from meiotic to mitotic chromosome segregation48. Our results indicated that the PN arrest specific down regulated genes mainly related to these

biological process. For example, the cyclin associated kinase (_CCNA2_ and _CCND3_) were required for sister chromatid segregation49, and structural maintenance and segregation of chromosome

proteins (_SMC3_ and _BUB1B_) have been reported to be in associated with developmental potential of human pre-implantation zygotes50. Cell cycle related genes (_CDC7_, _CDC23_ and

_CDC25A_) and some other genes including _DBF4, YWHAB, SKP2 and MDM2_ were also found to be significantly down regulated in PN arrest group. Moreover, some other genes specifically

down-regulated in PN arrest zygotes, including check point proteins codon genes (_RAD54B_ and _RAD51_)51,52, DNA repair related genes (_XRCC3_)53, chromosome integrity maintenance genes

(_NBN_)54,55 and _SHFM1_, all of which were involved in key proteins for homologous recombination. In addition, we also found some genes down regulated overlap for both Poly-PN and PN arrest

groups and these genes in different annotations were classified according to the expression specification and illustrated in a model (Fig. 5). Taken together, our work found Poly-PN have

some problems in oocyte meiosis and RNA processing, whereas PN arrest showed defects during mitosis cell cycle or homologous recombination during meiosis and this could provide new targets

for therapeutic intervention by modulating these corresponding signaling pathways in the future. Remarkably, as the scarce of the RTFF patients, we could not collect enough oocyte samples

for single cell RNA sequencing. So more clinical cases need to be collected and further verification need to be performed in the future. METHODS ETHICS STATEMENT All procedures were approved

by the Research Ethics Committee of Shanghai Jiao tong University School of Medicine and informed consent was obtained from participants at IVF center of the Ninth people’s hospital. We

confirmed that all patients have written informed consent for the use of their zygotes for this research. Animals were maintained at 23 °C in a 12-h (7:00–19:00) light and 12-h (19:00–7:00)

dark schedule, and all experimental procedures were performed in accordance with Institutional Animal Care and Use Committee guidelines of Shanghai Jiao Tong University School of Medicine.

PATIENTS, OVARIAN STIMULATION, OOCYTE RETRIEVAL, AND THE IVF/ICSI PROCEDURE For all patients in our study, five types of ovarian stimulation protocols were used: (1) Human Menotrophins

Gonadotrophin (hmG, Lizhu Pharmaceutical Trading Co.) co-treated with Medroxyprogesterone acetate (MPA, Shanghai Sine Pharmaceutical Ltd.) (hMG + MPA); (2) Human Menotrophins Gonadotrophin

co-treated with Clomifene Citrate (CC, Medochemie Ltd.) (hMG + CC); (3) Human Menotrophins Gonadotrophin co-treated with Medroxyprogesterone acetate and Clomifene Citrate (hMG + MPA + CC);

(4) Human Menotrophins Gonadotrophin co-treated with Medroxyprogesterone acetate and ethinyl estradiol (EE, Shanghai Sine Pharmaceutical Ltd.) (hMG + MPA + EE) and (5) Short protocol, in

which patients were administered with GnRHa daily beginning on menstrual cycle day 2 and with hMG daily beginning on menstrual cycle day 3. Follicle growth was monitored by ultrasound

examination. Serum FSH, LH, E2, and progesterone concentrations were measured serially using the chemiluminescence (Abbott Biologicals B.V.) method on the same days as the ultrasound exams.

Human Chorionic Gonadotrophin (hCG, Lizhu Pharmaceutical Trading Co.) at a 1000–5000 IU dose was administered when the dominant follicles reached 18 mm in diameter. Cumulus oocyte complexes

were recovered transvaginally with ultrasound guidance 34–36 hours post hCG. After retrieval, oocytes were maintained in human tubal fluid (HTF; Irvine Scientific) medium plus 10% synthetic

serum substitute (SSS; Irvine Scientific) for about 2 hours before _In vitro_ fertilization (IVF)/Intracytoplasmic sperm injection (ICSI). For ICSI treatment, the cumulus oophorus were

removed mechanically from oocytes with denuding pipettes in solution with 80IU hyaluronidase (Sigma) followed by injection. For IVF treatment, cumulus oocyte complexes were inseminated with

about 0.3–0.5 × 106/ml motile spermatozoa in HTF medium and the cumulus oophorus were removed 18 hours later. Fertilized eggs from both IVF and ICSI groups were cultured in 20 μl continuous

single culture medium (CSC: Irvine Scientic: USA) individually under oil and incubated at 37 °C humidified atmosphere under 5% CO2, 5% O2, and 90% N2 for pre-implantation culture. As a

policy of our center, fertilization was assessed by the presence of two pronuclei 16–18 hours post insemination, followed by confirming the embryonic development 66–68 hours post

insemination. The zygotes with more than three tiny pronuclei following the ICSI procedure were recognized as Poly-PN zygotes. The zygotes with normal pronuclei but failed to fuse until

66–68 hours post fertilization were name as PN-arrest zygotes. Tri-PN zygotes from four different IVF patients were used as controls. All samples above were collected and vitrified using

Cryotip method and then stored in liquid nitrogen until subsequent experimental treatment. PREPARATION AND QUALITY CONTROL OF SINGLE-CELL CDNAS The method for RNA extraction was carried out

as described previously56. Briefly, after thawing, each zygote was washed twice and transferred into lysate buffer. Then the reverse transcription reaction was performed directly on whole

cell lysate using SuperScript II reverse transcriptase (Life Technologies). We performed 15 cycles of PCR to amplify cDNA and the PCR product was purified by using AMPure XP beads (Beckman

Coulter). Agilent high-sensitivity DNA chip kit on a BioAnalyzer (Agilent Technologies) was used for checking the quality of cDNAs according the size distribution to ensure cDNAs contained

few short fragments (<500 bp) and showed peak sizes between 1.5 kb–2 kb. RNA-SEQ LIBRARY CONSTRUCTION AND SEQUENCING According to the manual of TruePrep DNA Library Prep Kit V2 for

Illumina (Vazyme Biotech), the quality of RNA-Seq sequencing library was checked by using Agilent high-sensitivity DNA chip. The libraries showing the peak around 300 bp was chosen for

high-throughput sequencing on the Illumina HiSeq 2500 platform using the dual index sequencing strategy with single-end reads length of 50 bp. BIOINFORMATICS PROCESS FOR SEQUENCING DATA

Individual sample from different zygotes has its own unique barcode sequence and could be separated from clean data. We used Tophat v2.0.957 to assemble the reads into NCBI build 37 hg19

genome and used Cufflinks v2.1.158 to calculate gene expression level. Clustering was used to process hierarchical clustering using Euclidean distance metric in the R packages59. Gene

expression levels were measured by using fragment per kilobase of exon per million mapped reads (FPKM). To rule out technical errors and increase the power to detect biological function, all

reference genes with average FPKM > 0.5 in any of three groups and the criterion of _P_ < 0.001 or _P_ < 0.01 together with FC (fold change) >2 or <0.5 were used to identify

differentially expressed genes for subsequent biological analysis using ArrayTrackTM software (FDA’s own bioinformatics and genomics tool,

http://www.fda.gov/ScienceResearch/BioinformaticsTools/Arraytrack/default.htm). KEGG PATHWAY ANALYSIS Database for Annotation Visualization and Integrated Discovery (DAVID V6.7;

https://david.ncifcrf.gov/) was used to perform KEGG pathway analysis60,61. SHRNA DESIGN AND _IN VITRO_ TRANSCRIPTION For short hairpin RNA (shRNA) design, we selected an siRNA-target

sequence on the NCBI RNAi database for each targeted genes, and the forward and reverse primers for each gene (SKP1 F: ATAGGGGGCT GCAAACTACT TAGACATTTC AAGAGA ATGT CTAAGTAGTT TGCAGCCTTT

TTTG; SKP1 R: GATCCAAAAA AGGCTGCAAA CTACTTAGAC ATTCTCTTGA AATGTCTAAG TAGTTTGCAG CCCC; PPP2CA F: ATAGGGTGGA ACTTGACGAC ACTCTTATTC AAGAGATAAG AGTGTCGTCA AGTTCCATTT TTTGPPP2CA R: GATCCAAAAA

ATGGAACTTG ACGACACTCT TATCTCTTGA ATAAGAGTGT CGTCAAGTTC CACC) were annealed and cloned into a T7 promoter containing vector pcDNA3.1(+) using Bsa1 and BamH1 restriction enzyme site, shRNA was

transcribed _in vitro_ from linearized pcDNA3.1-shRNA plasmid using MEGA short script T7 kit (Life Technology) and purified using MEGA clear kit (Life Technology) and mixed in RNase-free

water at the concentration of 50 ng/μl for subsequent use. OOCYTE MICROINJECTION, PARTHENOGENETIC ACTIVATION AND DEVELOPMENT ASSESSMENT Female mice aged 6–8 weeks were induced to

superovulate by i.p. injection of 10 IU of pregnant mare’s gonadotrophin (PMSG) (Ningbo Hormone Products Co.). Cumulus oocyte complexes (COCs) were collected at 46 h post PMSG. For COCs

retrieval, the ovaries were removed immediately and put into 4 ml HTF medium plus 10% SSS (Irvine Scientific) and 0.2 mM IBMX (Sigma). The COCs were released into this medium by puncturing

ovaries with a 27 g needle. The cumulus cells were released mechanically using mouth pipette and only those with normal morphologies were used for RNA injection. All injected oocytes were

cultured for maturation in a CO2 incubator for 16 hours for maturation before activation. The activation medium used was KSOM (Millipore) supplemented with 10 mM SrCl2. After being washed

twice in activation medium, oocytes were incubated first in activation medium for 2.5 hours and then in activation medium without SrCl2 for 3.5 hours at 37 °C in a humidified atmosphere with

5% CO2, 5% O2, and 90% N2. Both the activation medium and KSOM for subsequent short culture of oocytes were supplemented with 5 μg/mL cytochalasin B. Six hours after the onset of

activation, the fertilization rate was assessed by count pronuclear formation. STATISTICAL ANALYSIS Serum hormone data were analyzed by GraphPad Prism software using two-way repeated

measures ANOVA. Bonferroni post tests were used for pairwise comparisons. **_P_ < 0.01; ***_P_ < 0.001. Data for relative expression levels in Poly-PN and PN arrest zygotes were

separately compared with control were analyzed using a two-tailed, unpaired Student’s t test. _P_ < 0.001 indicated as significantly different. The maturation and fertilization rate of

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4, 44–57 (2009). Article  Google Scholar  Download references ACKNOWLEDGEMENTS We thank the voluntary research participants and all the doctors and embryologists in our center. We also

thank Qiang Wu (SJTU) for technique assistance for RNA sequencing and Aaron J. Hsueh (Stanford) for critical reading of manuscript. This work was supported by grants from the National

Natural Science Foundation of China (Grant No. 31200825, 81571397 and 81571486), the Fundamental Research Funds for the Central Universities (17JCYB12), Shanghai Committee of Science and

Technology, China (Grant No. 16411963800) and Shanghai Three-year Plan on Promoting TCM Development, China (Grant No. ZY3-LCPT-2-2006). AUTHOR INFORMATION Author notes * Lun Suo and Yu xiao

Zhou contributed equally. AUTHORS AND AFFILIATIONS * Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

Lun Suo, Hai bo Wu, Qi feng Lyu, Li hua Sun & Yan ping Kuang * Center for Comparative Biomedicine, MOE Key Laboratory of Systems Biomedicine, Institute of Systems Biomedicine, SCSB,

Shanghai Jiao Tong University (SJTU), Shanghai, 200240, China Yu xiao Zhou, Li ling Jia & Jin Zheng * Department of Genetics, School of Medicine, Stanford University, Stanford, CA,

94305, USA Han Sun Authors * Lun Suo View author publications You can also search for this author inPubMed Google Scholar * Yu xiao Zhou View author publications You can also search for this

author inPubMed Google Scholar * Li ling Jia View author publications You can also search for this author inPubMed Google Scholar * Hai bo Wu View author publications You can also search

for this author inPubMed Google Scholar * Jin Zheng View author publications You can also search for this author inPubMed Google Scholar * Qi feng Lyu View author publications You can also

search for this author inPubMed Google Scholar * Li hua Sun View author publications You can also search for this author inPubMed Google Scholar * Han Sun View author publications You can

also search for this author inPubMed Google Scholar * Yan ping Kuang View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS L.S., Y.X.Z. and

Y.P.K. designed the study; L.S., Q.F.L. and L.H.S. collected the samples; Y.X.Z., L.L.J. and J.Z. constructed the library; L.S. and H.B.W. performed the RNAi experiment; L.S., Y.X.Z. and

H.S. analyzed the data; L.S. and Y.P.K. supervised the study; L.S. wrote the manuscripts. CORRESPONDING AUTHORS Correspondence to Lun Suo or Yan ping Kuang. ETHICS DECLARATIONS COMPETING

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Transcriptome profiling of human oocytes experiencing recurrent total fertilization failure. _Sci Rep_ 8, 17890 (2018). https://doi.org/10.1038/s41598-018-36275-6 Download citation *

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