High-throughput sperm differential proteomics suggests that epigenetic alterations contribute to failed assisted reproduction.
OBJECTIVE
Are there quantitative alterations in the proteome of normozoospermic sperm samples that are able to complete IVF but whose female partner does not achieve pregnancy?


CONCLUSIONS
Normozoospermic sperm samples with different IVF outcomes (pregnancy versus no pregnancy) differed in the levels of at least 66 proteins.


BACKGROUND
The analysis of the proteome of sperm samples with distinct fertilization capacity using low-throughput proteomic techniques resulted in the detection of a few differential proteins. Current high-throughput mass spectrometry approaches allow the identification and quantification of a substantially higher number of proteins.


METHODS
This was a case-control study including 31 men with normozoospermic sperm and their partners who underwent IVF with successful fertilization recruited between 2007 and 2008.


METHODS
Normozoospermic sperm samples from 15 men whose female partners did not achieve pregnancy after IVF (no pregnancy) and 16 men from couples that did achieve pregnancy after IVF (pregnancy) were included in this study. To perform the differential proteomic experiments, 10 no pregnancy samples and 10 pregnancy samples were separately pooled and subsequently used for tandem mass tags (TMT) protein labelling, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, liquid chromatography tandem mass spectrometry (LC-MS/MS) identification and peak intensity relative protein quantification. Bioinformatic analyses were performed using UniProt Knowledgebase, DAVID and Reactome. Individual samples (n = 5 no pregnancy samples; n = 6 pregnancy samples) and aliquots from the above TMT pools were used for western blotting.


RESULTS
By using TMT labelling and LC-MS/MS, we have detected 31 proteins present at lower abundance (ratio no pregnancy/pregnancy < 0.67) and 35 at higher abundance (ratio no pregnancy/pregnancy > 1.5) in the no pregnancy group. Bioinformatic analyses showed that the proteins with differing abundance are involved in chromatin assembly and lipoprotein metabolism (P values < 0.05). In addition, the differential abundance of one of the proteins (SRSF protein kinase 1) was further validated by western blotting using independent samples (P value < 0.01).


CONCLUSIONS
For individual samples the amount of recovered sperm not used for IVF was low and in most of the cases insufficient for MS analysis, therefore pools of samples had to be used to this end.


CONCLUSIONS
Alterations in the proteins involved in chromatin assembly and metabolism may result in epigenetic errors during spermatogenesis, leading to inaccurate sperm epigenetic signatures, which could ultimately prevent embryonic development. These sperm proteins may thus possibly have clinical relevance.


BACKGROUND
This work was supported by the Spanish Ministry of Economy and Competitiveness (Ministerio de Economia y Competividad; FEDER BFU 2009-07118 and PI13/00699) and Fundación Salud 2000 SERONO13-015. There are no competing interests to declare.
Human Genetics Research Group, IDIBAPS, Faculty of Medicine, University of Barcelona, Casanova 143, Barcelona 08036, Spain.
Rubén
R
Azpiazu
Alexandra
A
Amaral
Judit
J
Castillo
Josep Maria
JM
Estanyol
Marta
M
Guimerà
Josep Lluís
JL
Ballescà
Juan
J
Balasch
Rafael
R
Oliva
D000328:Adult; D044127:Epigenesis, Genetic; D005260:Female; D005307:Fertilization in Vitro; D006801:Humans; D008297:Male; D011247:Pregnancy; D040901:Proteomics; D013094:Spermatozoa / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D017211:Treatment Failure
assisted reproduction; differential proteomics; epigenetics; human sperm; isotopic tandem mass tags