Non-invasive gamete and embryo assessment is considered an important focus in assisted reproductive technologies (ART). Currently, the selection of embryos for transfer is based on morphological ...indices. Though successful, the field of ART would benefit from a non-invasive quantitative method of viability determination. Omics technologies, including transcriptomics, proteomics and metabolomics, have already begun providing evidence that viable gametes and embryos possess unique molecular profiles with potential biomarkers that can be utilized for developmental and/or viability selection. Unlike the human genome that is relatively fixed and steady throughout the human body, the human proteome, estimated at over a million proteins, is more complex, diverse and dynamic. It is the proteins themselves that contribute to the physiological homeostasis in any cell or tissue. Of particular interest in ART is the secretome, those proteins that are produced within the embryo and secreted into the surrounding environment. Defining the human embryonic secretome has the potential to expand our knowledge of embryonic cellular processes, including the complex dialogue between the developing embryo and its maternal environment, and may also assist in identifying those embryos with the highest implantation potential. Advances in proteomic technologies have allowed the non-invasive profiling of the human embryonic secretome with ongoing research focused on correlation with outcome. From a clinical perspective, embryo selection based on morphological assessment and non-invasive analysis of the human embryonic secretome may improve IVF success and lead to routine single embryo transfers.
Chromosome testing strategies, such as preimplantation genetic testing for aneuploidy (PGT-A), improve initial IVF outcomes by avoiding unwitting transfer of aneuploid embryos in morphology-based ...selection practices. Newer technologies have revealed that some embryos may appear to have intermediate whole chromosome (or parts of a chromosome termed segmental) copy number results suggesting trophectoderm mosaicism. An embryo with a trophectoderm mosaic-range result may be the only option for transfer for some patients. Recent data suggest that such mosaic embryos can be transferred without added risk of abnormal birth outcomes but may be associated with increased implantation failure and miscarriage rates, with higher values of mosaicism appearing to be less favourable for producing good outcomes. In this Position Statement, we provide guidance to laboratories, clinics, clinicians and counsellors to assist in discussions on the utility and transfer of mosaic embryos.
The aim of this work was to examine the effect of 1,2-propanediol (PrOH) and type of cryopreservation procedure (slow freezing and vitrification) on oocyte physiology. METHODS: Intracellular calcium ...of mouse metaphase II (MII) oocytes was quantified by fluorescence microscopy. The effect of PrOH on cell physiology was further assessed through analysis of zona pellucida hardening and cellular integrity. Protein profiles of cryopreserved oocytes were generated by time-of-flight mass spectrometry (TOF-MS). RESULTS: PrOH caused a protracted increase in calcium, which was sufficient to induce zona pellucida hardening and cellular degeneration. Using ‘nominally calcium free’ media during PrOH exposure significantly reduced the detrimental effects. Proteomic analysis identified numerous up- and down-regulated proteins after slow freezing when compared with control and vitrified oocytes. CONCLUSIONS: Using such approaches to assess effects on cellular physiology is fundamental to improving assisted reproduction techniques (ART). This study demonstrates that PrOH causes a significant rise in intracellular calcium. Using calcium-free media significantly reduced the increase in calcium and the associated detrimental physiological effects, suggesting that calcium-free media should be used with PrOH. In addition, analysis of the oocyte proteome following cryopreservation revealed that slow freezing has a significant effect on protein expression. In contrast, vitrification had a minimal impact, indicating that it has a fundamental advantage for the cryopreservation of oocytes.
BACKGROUND
The aim of this study was to examine the effect of the cryopreservation procedure (slow freezing or vitrification) and cryoprotectants (1,2-propanediol or dimethylsulphoxide) on mouse ...blastocyst gene expression.
METHODS
Cultured mouse blastocysts were cryopreserved with different protocols. Following thawing/warming, total RNA from re-expanded blastocysts was isolated, amplified and then analyzed using mouse whole-genome microarrays.
RESULTS
Compared with non-cryopresevered control blastocysts, gene expression was only significantly altered by slow freezing. Slow freezing affected the expression of 115 genes (P < 0.05). Of these, 100 genes exhibited down-regulation and 15 genes were up-regulated. Gene ontology revealed that the majority of these genes are involved in protein metabolism, transcription, cell organization, signal transduction, intracellular transport, macromolecule biosynthesis and development. Neither of the vitrification treatment groups showed statistically different gene expression from the non-cryopreserved control embryos. Hierarchical cluster analysis, did however, reveal that vitrification using 1,2-propanediol could result in a gene expression profile closest to that of non-cryopreserved blastocysts.
CONCLUSIONS
Investigating the effects of cryopreservation on cellular biology, such as gene expression, is fundamental to improving techniques and protocols. This study demonstrates that of the cryopreservation regimens employed, slow freezing induced the most changes in gene expression compared with controls.
Purpose
In men, obesity may lead to poor semen parameters and reduced fertility. However, the causative links between obesity and male infertility are not totally clear, particularly on a molecular ...level. As such, we investigated how obesity modifies the human sperm proteome, to elucidate any important implications for fertility.
Methods
Sperm protein lysates from 5 men per treatment, classified as a healthy weight (body mass index (BMI) ≤ 25 kg/m
2
) or obese (BMI ≥ 30 kg/m
2
), were FASP digested, submitted to liquid chromatography tandem mass spectrometry, and compared by label-free quantification. Findings were confirmed for several proteins by qualitative immunofluorescence and a quantitative protein immunoassay.
Results
A total of 2034 proteins were confidently identified, with 24 proteins being significantly (
p
< 0.05) less abundant (fold change < 0.05) in the spermatozoa of obese men and 3 being more abundant (fold change > 1.5) compared with healthy weight controls. Proteins with altered abundance were involved in a variety of biological processes, including oxidative stress (GSS, NDUFS2, JAGN1, USP14, ADH5), inflammation (SUGT1, LTA4H), translation (EIF3F, EIF4A2, CSNK1G1), DNA damage repair (UBEA4), and sperm function (NAPA, RNPEP, BANF2).
Conclusion
These results suggest that oxidative stress and inflammation are closely tied to reproductive dysfunction in obese men. These processes likely impact protein translation and folding during spermatogenesis, leading to poor sperm function and subfertility. The observation of these changes in obese men with no overt andrological diagnosis further suggests that traditional clinical semen assessments fail to detect important biochemical changes in spermatozoa which may compromise fertility.
Purpose
The aim of this study was to evaluate the incidence of an inter-chromosomal effect (ICE) in blastocyst-stage embryos from carriers of balanced chromosome inversions.
Methods
Infertility ...patients (
n
= 52) with balanced inversions (
n
= 66 cycles), and maternal age-matched controls that concurrently cycled (
n
= 66), consented to an IVF cycle with preimplantation genetic testing for aneuploidy (PGT-A). Blastocyst-stage embryos underwent trophectoderm biopsy for PGT-A with only euploid blastocysts transferred in a subsequent frozen embryo transfer. Subtypes of inversions were included in aggregate: paracentric/pericentric, polymorphic/non-polymorphic, male/female carriers, and varying inversion sizes.
Results
The incidence of aneuploidy was not significantly higher for the inversion patients compared to the controls (inversion = 48.8% vs. control = 47.2% ns). Following euploid blastocyst transfer, there were excellent live birth outcomes.
Conclusions
Carriers of balanced chromosome inversions did not exhibit higher aneuploidy rates for chromosomes that were not involved in the inversion compared to maternal age-matched controls, signifying the absence of an inter-chromosomal effect for this data set. These results provide the largest investigation of blastocyst embryos regarding the debated existence of an ICE resulting from the presence of an inversion during meiosis. However, further studies are warranted to investigate an ICE among inversions subtypes that were outside the scope of this study.
BACKGROUND The high frequency of chromosomal abnormalities observed in human gametes and embryos is unlike that seen in other mammalian species and is of great clinical significance, leading to high ...rates of pregnancy loss, and live-born children with aneuploid syndromes. Although much is known concerning the aneuploidy rates of oocytes, cleavage stage embryos and fetuses during pregnancy, the chromosomal status of blastocysts has been relatively little investigated. METHODS A total of 158 good quality blastocysts were examined using micromanipulation, whole genome amplification and comparative genomic hybridization. RESULTS From the obtained data, it was evident that the aneuploidy rate (38.8%) is significantly lower for blastocysts than for embryos at earlier stages (51%). However, in many cases, chromosome errors, including monosomy, imbalance affecting the larger chromosomes and complex aneuploidy persisted to this final stage of preimplantation development. CONCLUSIONS This study represents the first attempt to gain a detailed insight into the extent and type of chromosome errors seen at the blastocyst stage, using a comprehensive molecular cytogenetic method. Our data suggest that the blastocyst stage does not represent an absolute selective barrier, and that the majority of aneuploid embryos are lost at the time of implantation or shortly thereafter.
STUDY QUESTION
When a chromosome aneuploidy is detected in the first polar body and a reciprocal loss or gain of the same chromosome is detected in the second polar body, is the resulting embryo ...usually aneuploid for that chromosome?
SUMMARY ANSWER
When reciprocal aneuploidy occurs in polar bodies, the resulting embryo is usually normal for that chromosome, indicating that premature separation of sister chromatids (PSSC)—not non-disjunction—likely occurred in meiosis I.
WHAT IS KNOWN ALREADY
Single-nucleotide polymorphism-based microarray analysis can be used to accurately determine the chromosomal status of polar bodies and embryos. Sometimes, the only abnormality found is a reciprocal gain or loss of one or two chromosomes in the two polar bodies. Prediction of the status of the resulting embryo in these cases is problematic.
STUDY DESIGN, SIZE, DURATION
Blinded microarray analysis of previously diagnosed aneuploid embryos that had reciprocal polar body aneuploidy.
MATERIALS, SETTING, METHODS
IVF cycles were performed between 2008 and 2011 in patients aged 40 ± 3 years (range 35–47 years) with an indication for polar body-based aneuploidy screening. Thirty-five aneuploid vitrified Day 3 embryos were warmed, cultured to Day 5 and biopsied for microarray analysis. Predictions were made for the ploidy status of the embryo if PSSC or non-disjunction had occurred. The signal intensity for the aneuploid chromosome in the first polar body was compared between those that resulted in euploid and aneuploid embryos.
MAIN RESULTS AND THE ROLE OF CHANCE
Among 34 embryos with evaluable results, 31 were euploid on re-analysis. Of 43 chromosomes that had reciprocal aneuploidy in the polar bodies, 41 were disomic in the embryo, indicating that PSSC was likely to have occurred 95% (95% confidence interval 85–99%) of the time. The log 2 ratio signal intensity from the chromosomes that underwent non-disjunction, resulting in unbalanced embryos, were outliers when compared with those that underwent PSSC.
LIMITATIONS, REASONS FOR CAUTION
Although most embryos with reciprocal aneuploid polar bodies were euploid, it is unknown whether they maintain equivalent reproductive potential when transferred. Further study is needed to determine whether these embryos should be re-biopsied and considered for transfer.
WIDER IMPLICATIONS OF THE FINDINGS
This study is consistent with increasing evidence that PSSC is the primary cause of meiosis I errors in embryos from women of advanced reproductive age. Clinicians should be cautious in interpreting results from polar body aneuploidy screening, especially when only the first polar body is tested.
STUDY FUNDING/COMPETING INTEREST(S)
None.
In vitro maturation (IVM) of mammalian oocytes does not support the same rates of embryo development or pregnancy when compared to oocytes that have matured in vivo. Therefore, environment has a ...significant influence on the oocyte's ability to complete maturation and acquire the mRNA and proteins required for successful fertilization and normal embryonic development. The aim of this study was to analyze the MII oocyte transcriptome between in vivo and in vitro conditions. Total RNA was extracted, processed and hybridized to the Affymetrix GeneChip
® Bovine Genome Array. Following normalization of the microarray data, analysis revealed 10 differentially expressed genes after IVM compared to in vivo matured controls, including
Aqp3,
Sept7,
Abhd4 and
Siah2 (
P
<
0.05). K-means cluster analysis coupled with associated gene ontology, identified several biological processes affected by IVM, including metabolism, energy pathways, cell organization and biogenesis, and cell growth and maintenance. Quantitative real-time PCR validated the microarray data and also revealed altered expression levels after IVM of specific putatively imprinted genes,
Igf2r,
Peg3 and
Snrpn (
P
<
0.05). Distinct IVM transcription patterns reflected the oocyte's response to its surrounding environment. Monitoring transcription levels of key oocyte maturation genes may subsequently assist in improving IVM success.
PDF
