Kristin Murphy

Comparative analysis of three sperm DNA damage assays and sperm nuclear protein content in couples undergoing assisted reproduction treatment

STUDY QUESTION Is there an association between sperm DNA damage, measured by three different assays, sperm nuclear protein content and clinical outcomes in assisted reproduction treatment (ART)? SUMMARY ANSWER Sperm DNA damage measured by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and the Comet assay were significantly associated with ART outcomes in our single institution study. WHAT IS KNOWN ALREADY Abnormal protamine expression is known to be associated with sperm DNA damage and male infertility. A number of studies have shown a significant relationship between sperm DNA damage and ART outcomes. To date, there are no large studies providing direct comparisons of DNA damage tests within the same study population. Thus, the prognostic value for each method remains unknown. STUDY DESIGN, SIZE, DURATION Cross-sectional study of 238 men from infertile couples undergoing ART at the University Center for Reproductive Medicine, Utah, USA, between April 2011 and March 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS Sperm from men undergoing ART were tested for DNA damage using the alkaline Comet assay, TUNEL and flow cytometric chromatin evaluation (FCCE) assays. Histone retention was analysed using the aniline blue staining method, whereas protamine content (proteins P1 and P2) and ratio were analysed using acid urea gel electrophoresis. The prognostic value of each sperm DNA test to predict clinical pregnancy was calculated. MAIN RESULTS AND THE ROLE OF CHANCE Histone retention was associated with sperm DNA damage (P < 0.001), reduced embryo quality (P = 0.005) and clinical pregnancies (P < 0.001). The mean percentage of sperm with DNA damage was significantly higher in sperm from non-pregnant couples compared with that from pregnant couples, as measured by TUNEL assay (15.04 ± 1.16% versus 8.79 ± 0.56%; P < 0.001) and alkaline Comet assay (72.79 ± 2.49% versus 55.86 ± 2.29%; P < 0.001). There was no association between clinical pregnancies and DNA fragmentation index measured by FCCE (12.97 ± 1.46 versus 14.93 ± 1.65; P = 0.379). Of the protamine parameters analysed, only the P1/P2 ratio was associated with sperm count (P = 0.013), mens age (P = 0.037), maturity (P = 0.049) and blastocyst quality (P = 0.012). Histone retention and sperm DNA damage measured by Comet and TUNEL assays were associated with fertilization rate (P < 0.05), embryo quality (P < 0.05) and implantation rate (P < 0.05). LIMITATIONS, REASONS FOR CAUTION A potential drawback of this study is that it is cross-sectional. Generally in such studies there is more than one variable that could cause the effect. Analysing sperm is one part of the equation; there are also a number of female factors that have the potential to influence ART outcomes. Therefore, given the large and well-established role of female factors in infertility, normal sperm DNA integrity and protamination do not necessarily ensure clinical pregnancy in ART. Thus, female factors can reduce the prognostic value of sperm DNA tests. Further, our use of native semen instead of prepared sperm may have iatrogenically increased the DNA damage. WIDER IMPLICATIONS OF THE FINDINGS Alteration in sperm nuclear protein affects sperm DNA integrity. Further, with the current dataset, TUNEL and Comet assays appeared more predictive of ART success than FCCE. STUDY FUNDING/COMPETING INTEREST(S) No personal or direct financial support has been received for any of this work. The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

Non-motile sperm cell separation using a spiral channel

Microfluidic sperm sorting has historically relied on sperm motility. However, a motility-based sperm separation technology will not work when viable, non-motile sperm need to be separated from other tissues as occurs when performing testicular sperm extraction (TESE) and microdissection testicular sperm extraction (mTESE) techniques. This work demonstrates the use of inertial microfluidics technology using spiral channels to separate sperm from blood cells. The separation method, which is label-free, does not rely on sperm motility for sorting. Basic principles of spiral channel separations were used to design a specific channel and flow parameters for separating non-motile sperm from blood. The spiral channels dimensions were: initial radius, 0.7 cm; final radius, 0.899 cm; channel width, 150 μm; channel height, 50 μm; turns of spiral, 4 turns; and space between channels, 310 μm. If sperm are modeled as a 5 μm sphere, inertial microfluidics theory suggests that the sperm could be focused and separated from red blood cells (RBCs). Channels to implement these features were validated in a series of experiments. Mixed samples of RBCs and sperm were used to test the sperm separation capability of the device with the sample injection flow rate ranging from 0.1–0.52 ml min−1. After running the sample through the spiral channel, the samples were collected from four outlets and were inspected using microscopy. The best results were obtained at a 0.52 ml min−1 flow rate and generated a concentration ratio of 81%, representing the percent of sperm collected from the two outer outlets. For the same conditions, 99% of RBCs were collected from the two inner wall outlets. Using a high speed scanner, we were able to observe the focusing of the RBCs and general focusing of the sperm. As the sperm are not a uniform shape, they did not focus in a tight band, but were collected in a general region of the channel. Nevertheless, the purification ratio for these sperm was sufficient to greatly enhance the likelihood of finding rare sperm in TESE/mTESE samples containing millions of blood cells. Sequentially processing of the samples in the system proved to further improve the ratio of sperm to blood cells.

Microfluidics: The future of microdissection TESE?

ABSTRACT Non-obstructive azoospermia (NOA) is a severe form of infertility accounting for 10% of infertile men. Microdissection testicular sperm extraction (microTESE) includes a set of clinical protocols from which viable sperm are collected from patients (suffering from NOA), for intracytoplasmic sperm injection (ICSI). Clinical protocols associated with the processing of a microTESE sample are inefficient and significantly reduce the success of obtaining a viable sperm population. In this review we highlight the sources of these inefficiencies and how these sources can possibly be removed by microfluidic technology and single-cell Raman spectroscopy.

How the Father Might Epigenetically Program the Risk for Developmental Origins of Health and Disease Effects in His Offspring

In addition to DNA, the sperm provides epigenetic factors to the embryo upon fertilization. DNA methylation, histone modifications, and small noncoding RNAs are critical for spermatogenesis and are present in mature sperm. Recent evidence suggests that a subset of these epigenetic modifications have roles beyond sperm function, providing transcriptional cues for early embryonic development. Numerous associations have been observed between paternal age, lifestyle, or chemical exposures and offspring health, with alterations seen in sperm epigenetic marks. These studies implicate epigenetic mechanisms in paternally inherited offspring phenotype. In this chapter, we outline the major epigenetic modifications established in sperm and explore possible modes of epigenetic inheritance from the sperm to the fertilized embryo. In addition, we provide evidence of environmental effects on the epigenetic state of paternal germ line and transmission of epigenetic alterations to offspring.

Epigenetic Changes in the Paternal Germline

Germ cells function to transmit genetic information from one generation to the next. In mammals, mature male germ cells are uniquely specialized in order to overcome the challenges associated with achieving fertilization within the female reproductive tract, and to provide a single chromosomal complement to offspring. Male germ cell development, including primordial germ cell and gonocyte development, and spermatogenesis, entails numerous epigenetic changes, such as DNA methylation, histone modification, histone variant incorporation, and small-RNA mediated processes, which promote meiosis and structural remodeling. Moreover, male germ cells must also be epigenetically reset, or reprogrammed, in order to establish a new generation. During embryonic development, primordial germ cells are cleared of somatically associated epigenetic marks, and new marks are established that are sex-specific as well as germline-specific. Additionally, after fertilization occurs, the paternal pronucleus is epigenetically reprogrammed in early zygotic stages, allowing for embryonic totipotency. While epigenetic reprogramming events are nearly genome-wide, some modifications are retained at select loci. Thus, it is possible that epigenetic information is transgenerationally inherited via the paternal germline. This chapter provides a comprehensive review on known epigenetic changes that occur during male germ cell development, as well as questions that remain unanswered in the field. Finally, clinical implications for altered epigenetic states are discussed.