World J Mens Health.  2020 Jan;38(1):103-114. 10.5534/wjmh.190034.

Proteomic Signatures in Spermatozoa Reveal the Role of Paternal Factors in Recurrent Pregnancy Loss

• Mohanty G
• Jena SR
• Nayak J
• Kar S
• Samanta L

• KMID: 2465415
• DOI: http://doi.org/10.5534/wjmh.190034

Abstract

PURPOSE
To identify the paternal factors responsible for aberrant embryo development leading to loss of foetus in recurrent pregnancy loss (RPL) through proteomic analysis of ejaculated spermatozoa.
MATERIALS AND METHODS
This prospective study consisted of male partners of RPL patients (n=16) experienced with two or more consecutive unexplained miscarriages and with no female factor abnormality as revealed by gynaecologic investigation including karyotyping and age matched fertile healthy volunteers (n=20). All samples were collected during 2013 to 2015 after getting institutional ethical approval and written consent from the participants. Seminal ejaculates were collected by masturbation after 2 to 3 days of sexual abstinence and analyzed according to World Health Organization 5th criteria 2010. Two-dimensional difference gel electrophoresis followed by mass spectrophotometric analysis was used to identify differentially expressed proteins (DEPs). Western blotting was used for validation of the key proteins.
RESULTS
The data identified 36 protein spots to be differentially expressed by more than 2-fold change with p<0.05 considered as significant. Matrix-assisted laser desorption/ionization time of flight/mass spectrometry identified GPx4, JIP4, ZN248 to be overexpressed while HSPA2, GSTM5, TF3C1, CC74A was underexpressed in RPL group. Western blot analysis confirmed the differential expression of key redox associated proteins GPx4 and HSPA2 in the RPL group. Functional analysis revealed the involvement of key biological processes that includes spermatogenesis, response to oxidative stress, protein folding and metabolic process.
CONCLUSIONS
The present study provides a snapshot of the altered protein expression levels consistent with the potential involvement of the sperm chromatin landscape in early embryonic development.

Keyword

Embryo loss; Paternal factors; Proteomics; Recurrent pregnancy loss; Spermatozoa; Two-dimensional difference gel electrophoresis

MeSH Terms

Abortion, Spontaneous
Biological Processes
Blotting, Western
Chromatin
Embryo Loss
Embryonic Development
Female
Healthy Volunteers
Humans
Karyotyping
Male
Masturbation
Metabolism
Oxidation-Reduction
Oxidative Stress
Pregnancy*
Prospective Studies
Protein Folding
Proteomics
Sexual Abstinence
Spectrum Analysis
Spermatogenesis
Spermatozoa*
Two-Dimensional Difference Gel Electrophoresis
World Health Organization
Chromatin

Figure

• 

  Fig. 1 Two-dimensional (2D)-difference gel electrophoresis of proteins isolated from sperm. (A) Control (labelled with Cy3). (B) Recurrent pregnancy loss group (labelled with Cy5). (C) Internal control (labelled with Cy2). (D) Overlay image. (E) Silver stained image showing the identification of 6 spots circled on the 2D gel and marked with their respective spot numbers as designated by DeCyder™ software.

• 

  Fig. 2 (A) Venn diagram showing the distribution pattern of the proteins identified through two-dimensional-difference gel electrophoresis. (B) Distribution pattern of differentially expressed proteins based on protein abundance. (C) Expression profile and densitometric analysis of two key proteins (GPx4 and HSPA2) in the spermatozoa of RPL patients compared to fertile donor. RPL: recurrent pregnancy loss, GAPDH: glyceraldehyde 3-phosphate dehydrogenase. *p<0.05 with respect to control.

• 

  Fig. 3 Schematic representation of proposed hypothesis of involvement of oxidative stress in spermatozoa as epigenetic regulator of paternal factors in recurrent pregnancy loss (RPL). GAPDH: glyceraldehyde 3-phosphate dehydrogenase, 2D-DIGE: two-dimensional difference gel electrophoresis, MS: mass spectrometry.

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