Sperm Phenotype and Differentially Methylated Regions

Study Overview
=================
Brief Summary
-----------------
Testicular dysgenesis syndrome (TDS) is known to cause epigenetic abnormalities in spermatozoa. Anogenital distance (AGD) is considered to be a suitable clinical marker of TDS, but the direct link between AGD and epigenetic abnormalities is still missing. Infertile men (n=10) presenting with shortened AGD and a control group of normal semen donors (n=10) with normal AGD will then be asked to provide one semen sample each. Using a flow cytometer and sorter (FACS) their spermatozoa will be sorted into populations of spermatozoa with/without DNA fragmentation or with/without chromatin decondensation. These sorted populations of spermatozoa will then be examined for differences in epigenetic imprinting differences using whole genome expression analysis. Whereas the sorting of spermatozoa will be carried out in Basel, the epigenetic analysis will be carried at the University of Geneva.

Detailed Description
-----------------
A subset of 10 men with shortened AGD (together with a control group of 10 fertile donors with normal AGD) will be asked to provide up to three semen samples, each of which then will be sorted with FACS into subpopulations with/without DNA fragmentation and into subpopulations with/without chromatin decondensation. Spermatozoa with fragmented DNA will be separated through FACS-sorting of spermatozoa with intact DNA using the YoPro 1-dye, which has been shown to correlate significantly with the degree of DNA fragmentation in the nuclei of sperm. In addition, spermatozoa with abnormal chromatin remodelling will be separated through sorting from spermatozoa with condensed chromatin using the fluorochrome chromomycin A3 (CMA3), which competes for protamin for binding to the minor groove of DNA thereby correlating with the persistence of histones in the sperm nuclei. Pilot experiments have demonstrated the highly significant and close correlation of CMA3 with anilin blue staining. Anilin blue staining is not suitable for the sorting experiment, because it requires fixation of the spermatozoa. Sorting based on CMA3 can be carried out with living spermatozoa. The sorted and anonymized samples will then be sent frozen in dry ice to a laboratory at the University of Geneva for the assessment of differences in the epigenetic imprinting of the DNA using whole genome expression studies.

Official Title
-----------------
Association Between Anogenital Distance, Sperm Phenotype and Epigenetics in Infertile Men.

Conditions
-----------------
Infertility, Male

Intervention / Treatment
-----------------
* Diagnostic Test: obtention of up to three semen samples


Participation Criteria
=================
Eligibility Criteria
-----------------
Inclusion Criteria: infertile men with known anogenital distance Exclusion Criteria: sperm concentration must be more than 15 million/ml to allow appropriate sorting with flow cytometry...

Ages Eligible for Study
-----------------
Minimum Age: 20 Years
Maximum Age: 55 Years

Sexes Eligible for Study
-----------------
Male

Study Plan
=================
How is the study designed?
-----------------

Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| subfertile men<br>10 infertile men with shortened AGD (< 40 mm) | Diagnostic Test: obtention of up to three semen samples<br>* sorting of spermatozoa with flow cytometry. In the presence of insufficient numbers of spermatozoa after sorting (<15 mill), up to three semen samples will be collected.<br>* Other names: conventional semen analysis followed by sorting;|
| fertile semen donors<br>10 fertile semen donors with normal AGD (>40 mm) | Diagnostic Test: obtention of up to three semen samples<br>* sorting of spermatozoa with flow cytometry. In the presence of insufficient numbers of spermatozoa after sorting (<15 mill), up to three semen samples will be collected.<br>* Other names: conventional semen analysis followed by sorting;|

What is the study measuring?
-----------------
Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| anogenital distance and epigenetics | number of differentially methylated transposable regulatory sequences in the genome of sorted spermatozoa. | 6 months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| sperm phenotype 1 | based on conventional semen analysis: concentration of spermatozoa (in mill/ml). | 6 months | 
| sperm phenotype 2 | based on conventional semen analysis: progressive motility (in %). | 6 months | 
| sperm phenotype 3 | based on conventional semen analysis: normal morphology (in %), staining). | 6 months | 
| sperm phenotype 4 | chromatin decondensation (as given by % of CMA3 staining). | 6 months | 
| sperm phenotype 5 | DNA fragmentation (% of YoPro 1-staining). | 6 months |