Chromatin Protamination and Catsper Expression in Spermatozoa Predict Clinical Outcomes after Assisted Reproduction Programs

Abstract

Identification of parameters predicting assisted reproductive technologies (ARTs) success is a major goal of research in reproduction. Quality of gametes is essential to achieve good quality embryos and increase the success of ARTs. We evaluated two sperm parameters, chromatin maturity and expression of the sperm specific calcium channel CATSPER, in relation to ART outcomes in 206 couples undergoing ARTs. Chromatin maturity was evaluated by Chromomycin A3 (CMA3) for protamination and Aniline Blue (AB) for histone persistence and CATSPER expression by a flow cytometric method. CMA3 positivity and CATSPER expression significantly predicted the attainment of good quality embryos with an OR of 6.6 and 14.3 respectively, whereas AB staining was correlated with fertilization rate. In the subgroup of couples with women ≤35 years, CATSPER also predicted achievement of clinical pregnancy (OR = 4.4). Including CMA3, CATSPER and other parameters affecting ART outcomes (female age, female factor and number of MII oocytes), a model that resulted able to predict good embryo quality with high accuracy was developed. CMA3 staining and CATSPER expression may be considered two applicable tools to predict ART success and useful for couple counseling. This is the first study demonstrating a role of CATSPER expression in embryo development after ARTs programs.

Figure 1

Association between CMA3 or AB and ART outcomes. (A) upper panel: Examples of spermatozoa positive (a) or not (b) after staining with CMA3; middle panel: Box plot representing sperm CMA3 positivity in the two groups with embryos with A quality (EQ_A) <50% (n = 133) and ≥50% (n = 9); lower panel: Receiver Operating Characteristic curve of the ability of CMA3 positivity to predict the attainment of at least 50% EQ_A. (B) upper panel: Examples of spermatozoa positive (a) or not (b) after staining with AB; middle panel: Association between sperm AB positivity and FR. Note that statistical analyses have been performed using FR as a continuous variable, even if grouped in quartiles for graphical purposes; lower panel: Receiver Operating Characteristic curve of the ability of AB positivity to predict the attainment of at least 80% FR. *p ≤ 0.05; FR: Fertilization rate; EQ_A: embryos with A quality; AUC: Area under the curve; CI: confidence interval.

Figure 2

Association between CATSPER1 expression and embryo quality and pregnancy achievement. (A) Typical histogram of CATSPER1 fluorescence in spermatozoa showing overlay of test sample (open) and corresponding negative control (rabbit serum, grey). (B) Images obtained by fluorescence microscopy and relative bright field of spermatozoa stained with CATSPER1 antibody (left) or rabbit serum (right). (C) Box plot representing CATSPER1 expression in groups with embryo quality A (EQ_A) <50% (n = 120) and ≥50% (n = 16). (D) Receiver operating characteristic curve of the ability of CATSPER1 MFI to predict the attainment of at least 50% EQ_A. E: Box plot representing CATSPER1 expression in couples achieving (n = 20) or not (n = 45) clinical pregnancy in the subpopulation of couples with female partners ≤35 years. *p ≤ 0.05; AUC: Area under the curve; CI: confidence interval; MFI: median fluorescence intensity.

Figure 3

Predictive model of embryo quality. Receiver operating characteristic curve of the probability of obtaining a percentage of embryo quality of type A ≥50% as predicted by the proposed binary logistic regression model described in the text. AUC: area under the ROC curve; CI: confidence interval.