Several studies indicate that Nitric Oxide (NO) plays an important role in the physiology of the reproductive system in mammals. It has been shown that NO affects sperm motility, it regulates the tyrosine phosphorylation of different sperm proteins, it enhances the sperm binding ability to the zona pellucida and it modulates the acrosome reaction.

The enzyme responsible for NO synthesis, the Nitric Oxide Synthase (NOS), has also been identified in the oocytes, cumulus and corona cells, as well as in the oviduct. For these reasons, the NOS presence at the fertilization site could be a key element to determine the success of this process. Therefore, carrying out in vitro studies to better understand NO's role in the fertilization process, especially in human sperm capacitation, could improve the outcome of the Assisted Reproductive Techniques (ARTs) due to an improvement both in the diagnosis of infertility and in the prognosis of treatment success.

This study is carried out in collaboration with the Animal Physiology Department from the Veterinary Faculty (University of Murcia, Spain) and it is funded by the European Commission under the Horizon 2020 Programme.

Main Objective:

Determine changes in the NOS expression and protein nitrosylation in fertile and infertile patients, when adding L-Arginine, the substrate for NO synthesis, to the sperm capacitation medium in the presence/absence of human follicular fluid (hFF).

Secondary Objectives:

1. Investigate if the effects of hFF on sperm capacitation are associated to NO levels in the fluid.

2. Identify the correlation between NO levels in hFF and the clinical outcomes from ARTs.

   METHODOLOGY

   A. Population The population included in the study will comprise both semen donors and couples attending the clinic IVI Murcia for a treatment of assisted reproduction via techniques of in vitro fertilization.

   B. Inclusion / exclusion criteria Depending on the objectives, both the population and inclusion and exclusion criteria may vary.

   Inclusion criteria Main Objective

   To examine the modifications in the NOS expression as well as the protein nitrosylation in fertile and subfertile patients. For this purpose, the following groups will be recruited:

   • a group of fertile males, such as the semen donors attending the clinic IVI Murcia;
   • a group of subfertile males composed of patients, who attend the clinic IVI Murcia to undergo an in vitro fertilization treatment and who possess one altered spermiogram parameter, according to the WHO (2010) guidelines.

   In order to reach the Secondary Objectives, couples who undergo an IVF or ICSI treatment will be included.

   Exclusion criteria Will be excluded couples who undergo preimplantation genetic diagnosis, were diagnosed with repetitive abortion (three or more consecutive abortions before week 20) or implantation failure (lack of pregnancy after three embryo transfers with good quality embryos in women under 36 years of age or after two embryo transfers in women over 36 years of age), as well as women over 40 years of age.

   C. Sample size:

   Since reference values for NOS expression are not available and since the variability of the distribution of progressive spermatozoa is high, it has been determined that to detect a difference of 20% with respect to the mean in a bilateral test, with a level of significance of 5% and 80% of statistical power, 41 semen samples per group will be necessary. A period of 24 months is estimated to reach the number of seminal samples.

   D. Experimental design and interventions Biomedical research study with control group (fertile semen donors) and experimental group (subfertile males)

   Interventions:

   Semen samples: will be obtained by masturbation after 2-7 days of sexual abstinence. All samples will be analyzed according to WHO guidelines (2010). To avoid unnecessary visits for the men included in the study, the semen sample will be requested during one of their visits to the clinic.

   Spermatozoa will be capacitated in the presence and absence of hFF. In a first set of experiments, the hFF will be supplemented with L-Arginine and the NOS inhibitor L-NAME. In another set of experiments the same supplementations to the capacitation medium will be examined, but without using hFF.

   A series of techniques such as Western blot, in situ nitrosylation and Biotin Switch Assay will be used to determine any changes in the NOS expression and protein nitrosylation in spermatozoa. The Western blot technique will also be applied to evaluate other capacitation markers such as the Protein Kinase A (PKA) activity, tyrosine phosphorylation and tyrosine nitration. The same treatment will be performed on all semen samples regardless of whether they come from patients or from semen donors. IN NO CASE WILL THESE SEMEN SAMPLES BE USED TO PERFORM AN ASSISTED REPRODUCTION TREATMENT.

   Human follicular fluid samples:

   To study the correlation between the NO concentration in follicular fluid and the success of the assisted reproduction treatment, the follicular fluid samples will be obtained during the oocyte pick up procedure. After oocyte removal, these fluids are normally eliminated, but in this case the fluids will be centrifuged for 10 minutes at 2000g and stored at -20°C until evaluation.

   The NO concentration will be determined with the help of a commercially available measurement system. NO is rapidly oxidized into two stable ions, namely nitrite and nitrate, which can be assayed by using ions selective electrodes. These electrodes have been miniaturized to be able to measure samples down to 30-40 µl with detection limits down to the low µM range for both species.

   The Western blot and protein nitrosylation assays will be performed inside the Animal Physiology laboratory, at the University of Murcia (Faculty of Veterinary Medicine).

   It should be noted that the only objective of analyzing the follicular fluid is to investigate after the assisted reproduction treatment if there is any correlation between the result of the treatment and the NO levels. Under no circumstances will embryo transfer decisions be made based on the NO levels in the follicular fluid.

   E. Collection of biological samples Sperm and follicular fluid samples will be evaluated in the present study.

   • Purpose of sample collection The samples collected in the present study will only be used for the completion of the research project. To each biological sample a code will be assigned, which will be independent from the NHC code (number of clinical history) of the patients and will only be known by the research team. Dr. Juan Carlos Martínez Soto will be responsible for the storage and safekeeping of the biological samples.
   • Method for sample collection The biological samples will only be obtained once the patient has signed the corresponding informed consent. The seminal samples will be obtained by masturbation after 2-7 days of sexual abstinence. To avoid unnecessary displacements, the sample collection will take place during the visit of the patient to the clinic.

   The follicular fluid samples will be obtained on the day of the oocyte pick up procedure performed for the in vitro fertilization treatment that the patients will follow. This fluid, which is normally discarded, will be used only to determine NO values.

   Obtaining this follicular fluid does not pose any additional risk or discomfort to those caused by the reproduction treatment, since the surgical procedure of oocyte pick up is used to obtain the follicular fluid.

   • Method for sample identification The semen and follicular fluid samples will be identified in a way which will allow to safeguard the identity of the participants.
   • Sample conservation Both seminal and follicular fluid samples will be stored in freezers in the laboratory of Andrology (IVI Murcia) at -80°C until further processing, under the supervision of Dr Juan Carlos Martínez Soto. The samples sent to the University of Murcia will be stored by Dr. Carmen Matás Parra. Both the facilities of the University of Murcia and those of the IVI clinic have a restricted access, therefore the samples will be always guarded. Upon completion of the study (January 2019), the surplus samples will be destroyed following the usual protocols of the clinic.

   F. Database The data regarding sample analysis (follicular fluid and semen samples), will be stored in Excel tables in the facilities of IVI Murcia. The data regarding the results of the assisted reproduction treatments that have been followed by the patients included in the study will be obtained from the IVI management system (SIVIS).

   G. Study variables

   Four classes of variables will be distinguished according to their relevance within the study:

   Primary Variable: NO levels.

   Secondary Variables: NOS expression, rate of progressive sperm, pregnancy rate, biochemical pregnancy, pregnancy evolution, implantation rate, ectopic pregnancy rate.

   Control Variables: Tyrosine phosphorylation (optical density units), PKA (optical density units), protein nitrosylation (optical density units), nitrates and nitrites (micromolar units).

   Descriptive variables: maternal age, paternal age, male etiology, female etiology, BMI, smoking habit.

   H. Data analysis

   Descriptive analysis:

   Statistical summary of the data collected in the study. Categorical data will be presented in frequency tables and histograms. The continuous values will be summarized with the mean, standard deviation and confidence interval, they will be represented by graphs of quartiles or densities.

   The exploratory analysis of the data will allow to assess the quality of the data and the detection of anomalies.

   Analysis of homogeneity:

   The means and proportions of the descriptive variables (baseline and demographic) will be compared to validate the degree of comparability of the study groups. S chi-square test will be applied in the categorical variables and, assuming the data follow a normal distribution, a T-test in the continuous variables. If the data does not follow a normal distribution, a Mann-Whitney test would be applied.

   In the case of finding descriptive variables distributed in a way that there are statistical differences between the groups, they will be included in a regression model, to control their interference in the main relation to be studied.

   Comparison of NO expression:

   To validate a hypothesis of correlation of proportions in the NO expression, a Fisher test will be applied.

   To evaluate the effect of other covariables that can modify the behavior of NO expression, a logistic regression model will be defined.

   As for the comparison of protein nitrosylation, a Student's t-test will be applied assuming that the data follow a normal distribution. If the normality assumption will not be met, verified by a Kolmogorov-Smirnof test, a non-parametric U-MannWhitney test would be applied.

   In the same way, a linear regression model will be defined that allows the evaluation of the effect of other correlated variables.

   Evaluation of seminal quality:

   For the set of variables that make up the seminal quality, a t-Student test will be applied assuming that the data follow a normal distribution. If the normality assumption will not be met, verified by a Kolmogorov-Smirnof test, a non-parametric U-MannWhitney test would be applied.

   Evaluation of gestational outcomes:

   For the set of variables that define the gestation results, a Fisher test will be applied.

   Workplan:

   Development stages and distribution of the tasks of the entire research team, and the assignments planned for the technical staff. Indicate in addition place / center of realization of the project.

   The design of the study, as well as the drafting of the project have been carried out by Doctors Carmen Matás Parra and Juan Carlos Martínez Soto.

   The team of gynecologists who make up the Unit of Reproductive Medicine at the IVI Murcia clinic, Dr. Jose Landeras, María Nicolás, Laura Fernández and Martina Trabalón, will be responsible for the inclusion of patients and semen donors in the study, as well as the direct responsible for the assisted reproduction treatment performed on patients. Such treatment includes: the choice of treatment to be performed, ovarian stimulation, oocyte pick up procedure to collect oocytes and follicular fluid, as well as embryo transfer.

   The embryologists Marta Molla, David Gumbao, Julián Marcos, Beatriz Amorocho and Ana Isabel Sanchez will be responsible for the process of oocyte fertilization in the fertilization laboratory, as well as the selection of embryos to be transferred.

   Florentin-Daniel Staicu, Carmen Matás Parra and Juan Carlos Martínez Soto will carry out the analysis of both seminal and follicular fluid samples. These analyses will be carried out both in the facilities of the IVI Murcia clinic and in the laboratories of the University of Murcia.

   Dr. Jorge Chavarro will conduct the epidemiological and biostatistical studies.

Inclusion Criteria:

• This study will include fertile males such as the semen donors attending the clinic IVI Murcia and a group of subfertile males composed of patients, who attend the clinic to undergo an in vitro fertilization treatment and who possess one altered spermiogram parameter (according to the WHO 2010 guidelines). These participants will be recruited to examine the modifications in the NOS expression as well as the protein nitrosylation in fertile and subfertile men.
• This study will also include couples who undergo an IVF or ICSI treatment, in order to investigate if the effects of human follicular fluid on sperm capacitation are associated to NO levels in the fluid and to identify the correlation between NO levels in the follicular fluid and the clinical outcomes from ARTs.

Exclusion Criteria:

• From this study will be excluded couples who undergo preimplantation genetic diagnosis, were diagnosed with repetitive abortion (three or more consecutive abortions before week 20) or implantation failure (lack of pregnancy after three embryo transfers with good quality embryos in women under 36 years of age or after two embryo transfers in women over 36 years of age), as well as women over 40 years of age.

This study will include: fertile and subfertile men (who will provide the semen samples) and women donors (who will provide the follicular fluid samples).

The IPD will not be shared at any point during this study. To preserve participant's anonymity, the biological samples (semen and follicular fluid) that will be collected will be identified with a code which will be different from the Number of Clinical History of the donors/patients.