Measuring Free Radicals in Human Sperm Cells Related to Microbiota and Lifestyle Factors

The cause of infertility can be due to a female factor or a male factor. In case of a male factor, it is often due to poor semen quality. However, the cause of poor quality is often unknown. In previous research, infertility problems in men were related to chemical processes in metabolism causing the formation of free radicals. Free radicals are physiological by-products of our body mechanisms. Free radicals are very reactive and can therefore react with a lot of molecules of cells within our body and cause damage. A balance between free radicals, which are also needed for physiological processes in the body, and antioxidants, which defuses the reactive free radicals, is most desirable. However, as stated in literature, there are a lot of factors that can influence extra free radical production, which causes overloading of the system, resulting in damage on cellular level. Free radicals in semen plasma and on the sperm cell could play a role in male infertility. Nonetheless, free radicals are not used as diagnostic markers due to the lack of detection systems, as free radicals are very short-lived. This study aims to introduce a new technique, called diamond magnetometry, to measure free radicals directly on the sperm cell and in serum. Diamond magnetometry involves very small diamond particles as magnetic sensors that engage a reaction with the free radicals on the sperm cell, causing signals that can be measured. To compare local free radical production with systemic free radical production, other diagnostic biomarkers are also measured in serum.

It is hypothesized that the composition of seminal microbiome could influence the free radical concentration. Therefore, this study also aims to explore the microbiota composition and see if this has an influence in semen quality and free radical production. At last, this study also want to correlate standard semen parameters (defined by the World Health Organisation), lifestyle factors and food intake, to detect a role for lifestyle in the production of free radicals.

To date, 15% of couples experience issues with fertility and in 40-50% of the cases it is thought to be due to fertility issues in men. Reactive oxygen species (ROS) and especially free radicals (the most reactive ones) play a key role in sperm capacitation and fertilisation. They are also believed to be a major contributor to male infertility. The free radical theory of infertility states that damage by free radicals is the main cause of infertility in males. However, they are difficult to measure due to their short lifetime and high reactivity.

Despite the relevance of free radicals, they are not used as diagnostic biomarkers nor as target for therapeutics since it is currently unknown where and when the free radicals are generated exactly, due to the insufficiency of the currently available techniques. Therefore, a new technique will be utilized called diamond magnetometry which allows nanoscale magnetic resonance measurements. This quantum sensing technique is uniquely sensitive and allows real-time single cell measurements with sub-cellular resolution. Unlike most other techniques this method is specific for free radicals. A first proof of principle experiment has been published in yeast cells and we have preliminary data which proofs that we can measure free radical generation in boar sperm. The concentration of free radicals will be related to standard semen-parameters according to the WHO guidelines, lifestyle factors and food intake and related with microbiota in semen plasma to explore possible correlations. Microbiota have been suggested to play a role in the context of reproduction. While several studies have been carried out focusing on the female reproductive system, less is known about male microbiota and its influence on fertility. In the past it was thought that the presence of bacteria in the semen was an indicator of infection. The next generation genetic-based approach has revealed that the human semen is not sterile and appear to host a specific microbiota. Alterations of the diversity of seminal microbiota or the abundance of specific bacteria have been associated with an altered morphology and motility of sperm cells. Nevertheless, the molecular processes through which bacteria are able to alter semen quality are unknown. Possible mechanisms may involve free radical damaging or alteration of other molecules (e.g. lipids peroxidation, DNA fragmentation) which may result in loss of fertilisation capability. Therefore, it is hypothesized that free radical generation is correlated to the diversity of microbiota and can be related to the semen-parameters defined by the WHO guidelines.

The primary objective of this study is to measure real-time free radicals in human single sperm cells using diamond magnetometry. This is correlated to the seminal microbiota composition to investigate the influence of the composition in free radical generation, and therefore a possible mechanism of microorganisms to alter semen quality. The free radical concentration is also related to other oxidative stress parameters in serum to compare local and systemic oxidative stress. At last, the free radical concentration will also be correlated to the semen analysis parameters (WHO guidelines), lifestyle factors and food intake to detect a role for lifestyle in free radical formation and semen quality.

The main study parameters will be the free radical concentration at sperm cells and serum, correlated with microbiota in seminal plasma and related to standard semen- parameters (WHO), malondialdehyde (MDA) and free thiols concentration in blood serum, lifestyle factors and food intake.

The patients are informed about the study at their intake at the Centre of Reproductive Medicine. After informed consent, patients will have to produce semen for evaluation in the context of standard care semen analysis. A sample of that ejaculate will be collected for the SIRIUS study to measure free radicals in. Next to that, blood will be drawn from the patients at the same day as the semen analysis for the free radical measurement in serum.

Free radical measurements will be performed using diamond magnetometry. Previously, diamond magnetometry was successfully used to quantify radical formation in the acrosome of boar sperm heads. This allowed quantification of radical formation locally in real time, during capacitation. This data is not published yet and is produced in the lab of Dr. Romana Schirhagl.

Semen plasma microbiota composition will be evaluated using quantitative polymerase chain reaction (qPCR) and 16S ribosomal ribonucleic acid (rRNA) sequencing methods using QIAGEN Sample and Assay Technologies.

MDA concentration in serum will be measured using Thio barbituric acid reactive substance (TBARS) assay. Free thiol concentration in serum will be measured using the Ellman technique. A questionnaire for lifestyle factors is implemented as part of standard care for the couples seeking fertility treatment. These outcomes and answers will also be used for SIRIUS.

For an indication of food intake of the subjects a food frequency questionnaire (FFQ) of Wageningen University will be used.

Eligibility Criteria:
Inclusion Criteria:

* Males of couples visiting the CRM at the UMCG between 18-55 years old.
* Planned semen-analysis as standard care.

Exclusion Criteria:

* Males who receive(d) chemo- and/or radiotherapy, use(d) testosterone suppletion and/or anabolic steroids
* Males who are azoospermic
* Males who have an abnormal SA due to genetic causes.
* Semen analysis with round cells \>2x106 /ml (as marker for infection)
* Males who currently use antibiotics