The study was a prospective case/control study. Patients (cases) and controls were recruited in the university hospitals of three French cities (Bordeaux, Marseille and Toulouse).
Cases were recruited when they came to the fertility centers for management of unexplained recurrent pregnancy loss (URPL).
Controls were recruited in the maternity departments of the same hospitals. They had proven fertility (full-term pregnancy with live birth, baby aged less than a year old when entering the study). They also had no history of ART treatment or RPL (i.e. no more than two recurrent miscarriages before 12 weeks of gestation).
Men were included in the case group if their couple had experienced more than two recurrent miscarriages before 12 weeks of gestation, with no female causes identified after a classic RPL check-up: anatomical causes (investigated by hysterosalpingography, hysteroscopy or 2D/3D ultrasound); endocrinological causes (measurement of TSH, anti-thyroid antibodies and FSH); autoimmune causes (measurement of anticardiolipin antibodies and lupus anticoagulant); thrombophilia (measurement of homocysteine, factor V Leiden, prothrombin and C-reactive protein). Infectious causes were not investigated unless the woman had chronic endometriosis, cervicitis or immunodepression. All pregnancies occurred naturally. The female partners were under 38 years old. Blood karyotypes were normal in both men and women. The men were healthy, with no identifiable cause of infertility, no family disease and no endocrine or immunological disorders. The controls included were age-matched ±3 years.
At inclusion, controls and cases were asked about fertility histories within the family and outside the couple, and the fertility history of their own couple (pregnancies, miscarriages, time to conceive). Surgical and medical histories, history of infections, professional exposure, and andrological conditions such as urinary infection, varicoceles, cryptorchidism and family histories of these conditions were investigated. Environmental factors were investigated by evaluating alcohol, caffeine and tobacco consumption, living environment and lifestyle. The male partner underwent clinical examination and a detailed questionnaire was completed on the female partner.
All participants were recruited on a voluntary basis and gave informed consent. This study was supported by a grant from the French Ministry of Health (PHRC N°11, 198 08, 2011, PARTHOM project). The sponsor had no role in the study. The project was approved by the institutional ethics review board (Comité de Protection des Personnes Sud Ouest et Outre-Mer II). This work was sponsored by the Universitary Hospital of Toulouse for regulatory and ethic submission.
Sperm samples were collected by masturbation after 3–6 days of sexual abstinence. Semen was allowed to liquefy for 30 min at 37 °C before analysis. Conventional semen analysis was performed according to WHO guidelines with similar methodology in the three laboratories. Sperm analysis is subject to external quality control in all centers. Ejaculate volume (vol, ml), sperm count (SC, 106 spermatozoa/ml), total sperm count (TSC = vol x SC, 106 spermatozoa/ejaculate), motility (percentage of rapidly progressive or slowly progressive spermatozoa), total motile sperm count (TMSC = TSC x motility, 106 spermatozoa/ejaculate) and vitality were assessed.
Sperm cell smears were prepared in each center for sperm morphology analysis. Morphology was studied according to David’s classification modified by Jouannet [18, 19]. We calculated the multiple anomalies index (MAI) which was the number of anomalies per abnormal spermatozoon. In order to reduce interoperator variability, sperm morphology analysis was performed blindly by a single technician in the Toulouse center.
The remaining semen sample was mixed with a cryoprotectant, frozen in straws within 1 h of collection and stored in liquid nitrogen according to the standard procedures used for sperm banking in the laboratories. All samples were stored in the Germethèque Biobank (BB-0033-00081, France) until further analysis centralized in Toulouse.
Sperm chromatin and DNA fragmentation exploration
Sperm chromatin condensation was evaluated in each center by aniline blue staining of semen samples fixed with 3% glutaraldehyde according to a previously published method .
Sperm DNA fragmentation index (DFI) and high DNA stainability (HDS) were measured by conventional sperm chromatin structure assay (SCSA) described by Evenson and Jost and routinely used in our laboratory as previously published .
DNA strand breaks were measured using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay according to a previously published method .
SCSA and TUNEL assay were centralized in the Toulouse laboratory. 5000 and 10,000 cells were analyzed by SCSA and TUNEL, respectively, by fluorescence-activated cell sorting (FACS) on a FC500 cytometer (Beckman Coulter Inc, Fullerton, CA, USA).
Sperm fluorescence in situ hybridization (FISH)
In the Toulouse center, cells were thawed and then washed twice with 5 ml PBS and centrifugation at 630 g. Samples were then fixed with fixation solution (acetic acid and methyl alcohol) for 30 min at 4 °C. After centrifugation at 1500 g, the supernatant was discarded and the pellet resuspended. 10 μl were dropped on a slide and cell density was verified by microscopy and adjusted accordingly. Slides were incubated for a minimum of 2 h at − 20 °C. DNA was decondensed by incubating slides in 1 M NaoH for 1 min, washed twice in SSC, then dehydrated in 70%, 90% and 100% ethyl alcohol baths (2 min each). Each slide was then incubated overnight with the different probes at 37 °C (Vysis probes (Abbott), CEP X spectrum green, CEP Y spectrum orange and CEP 18 spectrum aqua). After a 2 min wash in 2SSC 0.4% NP40 at 73 °C followed by a 1 min wash in 2SSC 03% NP40, slides were incubated with 1/2000e Hoechst for 3 min and washed for 3 min in PBS. Slides were finally mounted with Antifade mounting medium (Promega, Germany) and stocked at − 20 °C until read. Slides were analyzed under a Leica DM 6000 B microscope system. At least 5000 cells were read by a single reader for each slide.
Quantitative data were presented as median and interquartile range [q1–q3] due to the number of participants and as boxplots for graphic representation.
Standard non-parametric tests were used to compare cases and controls. For all quantitative data, the Mann-Whitney test was used. For qualitative data, differences between subgroups were tested with the Wilcoxon test and Fisher exact test. A non-parametric Spearman correlation was performed to analyze the relationships between various sperm parameters and FISH results, as well as sperm DNA fragmentation results and FISH results. All univariate and multivariate analyses were performed using SAS 9.3 software and the significance level was defined as 5%.
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.