Transcriptome profiling of human oocytes experiencing recurrent total fertilization failure
Transcriptome profiling of human oocytes experiencing recurrent total fertilization failure
Lun Suo 0
Yu xiao Zhou 1
Li ling Jia 1
Hai bo Wu 0
Jin Zheng 1
Qi feng Lyu 0
Li hua Sun 0
Han Sun 2
Yan ping Kuang 0
0 Department of Assisted Reproduction, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine , Shanghai , China
1 Center for Comparative Biomedicine, MOE Key Laboratory of Systems Biomedicine, Institute of Systems Biomedicine, SCSB, Shanghai Jiao Tong University (SJTU) , Shanghai, 200240 , China
2 Department of Genetics, School of Medicine, Stanford University , Stanford, CA, 94305 , USA. Lun Suo
OPEN 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.
Published; xx xx xxxx
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?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?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?29. The single cell sequencing technique has great potential in clinical implication30?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
Patient with recurred Poly-PN zygotes
Patient with recurred PN-arrest zygotes
HMG + MPA
HMG + CC
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),
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
SDEGs (Poly-PN/Control) N-Glycan biosynthesis
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.
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 THOC141, SF3B142, 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
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: (
) Human Menotrophins Gonadotrophin
(hmG, Lizhu Pharmaceutical Trading Co.) co-treated with Medroxyprogesterone acetate (MPA, Shanghai Sine
Pharmaceutical Ltd.) (hMG + MPA); (
) Human Menotrophins Gonadotrophin co-treated with Clomifene
Citrate (CC, Medochemie Ltd.) (hMG + CC); (
) Human Menotrophins Gonadotrophin co-treated with
Medroxyprogesterone acetate and Clomifene Citrate (hMG + MPA + CC); (
) Human Menotrophins
Gonadotrophin co-treated with Medroxyprogesterone acetate and ethinyl estradiol (EE, Shanghai Sine
Pharmaceutical Ltd.) (hMG + MPA + EE) and (
) 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 ?l
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 300bp 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/
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/?l 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 10mM 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 ?g/
mL cytochalasin B. Six hours after the onset of activation, the fertilization rate was assessed by count pronuclear
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 the oocytes between gene knock down and control group were analyzed by using
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).
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.
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-36275-6.
Competing Interests: The authors declare no competing interests.
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