Patients and control subjects
A cohort of nineteen patients with MMAF and their families were recruited for this study from the Affiliated Yantai Yuhuangding Hospital of Qingdao University. Twenty men with proven fertility, who already had at least one child, served as normal controls. The inclusion and exclusion criteria for this study was identical to those used in previously published reports [37]. Physical and andrological hormone examinations were also performed, and all of the selected participants exhibited normal external genitalia, bilateral testicular development, and normal andrological hormone levels. No abnormalities were observed in the bilateral spermatic veins upon palpation, and all patients presented with normal chromosomal karyotypes (46, XY). All participants answered a questionnaire about primary ciliary dyskinesia (PCD) -associated manifestations, including long instances of coughing with phlegm, year-round nasal congestion, chronic sinusitis, bronchiectasis, recurrent lower respiratory tract infections, pneumonia, and otitis media [38]. A total of 5 mL of peripheral blood was collected from each participant for whole genome and Sanger sequencing.
Whole-exome sequencing (WES) and Sanger sequencing validation
Whole-exome sequencing was performed as described previously [39]. The reads were aligned to the UCSC hg19 genome using the Burrows-Wheeler Aligner (http://biobwa.sourceforge.net/) and variants were annotated using ANNOVAR (http://www.openbioinformatics.org/annovar/). Each variant was then evaluated for pathogenicity using Polyphen2 (http://genetics.bwh.harvard.edu/pph2/) and Mutation Taster (http://www.mutationtaster.org/). They were also cross-referenced with the Exome Aggregation Consortium (ExAC) (http://exac.broadinstitute.org/), genome aggregation database (gnomAD, http://gnomad.broadinstitute.org/), 1000 Genomes Project (http://www.1000genomes.org/data), and ESP6500 (http://evs.gs.washington.edu/EVS/) databases for novelty and frequency analysis. Mutations that met these criteria were then assigned as missense, nonsense, frame-shift, or splice site variant and annotated as absent or rare variants. The DNAH1 mutations were further validated using Sanger sequencing and the primers used for this sequencing are listed in Supplementary Table 2.
Papanicolaou staining
Spermatozoa were examined using Papanicolaou staining in accordance with the World Health Organization standards for human semen examination and processing (5th ed.) with minor modifications to confirm any morphological changes in the sperm tails. Briefly, slides were fixed in 95% ethanol (Xilong, Shantou, China), and then immersed in an alcohol gradient from 80% to 50%. The slides were then rinsed with distilled water, and stained with haematoxylin (Zsbio, Beijing, China). Following this, the slides were dehydrated in an alcohol gradient from 50% to 90%, dyed with Orange G6 (Solarbio, Beijing, China) and EA50 (Solarbio, Beijing, China), and dehydrated again using 95% and 100% alcohol (Xilong, Shantou, China). Finally, the slides were washed in xylene and mounted using a permanent mounting medium.
Transmission electron microscopy (TEM)
Sperm samples were examined via TEM according to a previously published procedure that allows for the evaluation of subcellular structural changes in these samples [25]. Briefly, prepared spermatozoa were immobilised with 2.5% phosphate-buffered glutaraldehyde (Electron Microscopy Sciences, Hatfield, USA), then the samples were washed with 0.1 M phosphate buffer (pH 7.2) and post-fixed with 1% osmium tetroxide (Sigma-Aldrich, St. Louis, USA). Dehydration was performed by sequentially treating samples with an alcohol gradient and 100% acetone (Xilong, Shantou, China) and infiltrated with 1:1 acetone and resin (SPI Supplies, West Chester, USA). After infiltration, the samples were embedded and polymerised. Ultrathin sections (70 nm thick) were cut and collected on 200 mesh TEM copper grids, and then counterstained with uranyl acetate and lead citrate. The ultrastructure of each sample were observed and photographed using a Tecnai G2 Spirit transmission electron microscope (FEI, Oregon, USA) at 80 kV.
Immunostaining of spermatozoa
We performed immunostaining of the spermatozoa as described previously [18]. Briefly, the prepared spermatozoa were smeared onto poly L-lysine-coated slides, allowed to air-dry, washed in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde (PFA) (Sigma, St. Louis, USA) for 10 min at room temperature, and washed twice with PBS. Next, the samples were permeabilised using 0.2% Triton X-100 (Sigma, St. Louis, USA) and blocked for 30 min at room temperature. Slides were incubated with primary antibodies for 1 h at room temperature and then with secondary antibodies for 1 h at RT. Samples were subsequently washed three times with PBS and mounted using Vectorshield supplemented with DAPI (Vector Laboratories, Burlingame, USA), and examined under a laser scanning confocal immunofluorescence microscope (Carl Zeiss, Jena, Germany). The specific antibodies used in these assays are listed in Supplementary Table 3.
Ovarian stimulation, intracytoplasmic sperm injection, and embryo transfer
Ovulation was triggered in the wives of the patients using the long standard ovarian stimulation protocol. Briefly, women were given 75 IU Recombinant Follitropin Beta Injection (Merck Sharp & Dohme, Kenilworth, USA) and 75 IU human menopausal gonadotropin (Ferring Pharmaceuticals, Saint Prex, Switzerland), and their follicular response was evaluated after 5 days of gonadotrophin stimulation. Ultrasound was used to monitor the diameter of the follicles and when at least two follicles were ≥18 mm in diameter, patients received 6500 or 10 000 IU human chorionic gonadotropin (Livzon, Zhuhai, China). We retrieved oocytes 34–36 h later using vaginal ultrasound-guided follicular puncture.
The intracytoplasmic sperm injection (ICSI) procedure was performed as previously described [40]. Briefly, 18–19 h after fertilisation, samples were evaluated for the presence of two pronuclei and two polar bodies with fertilised oocytes individually cultured in G1 medium (Vitrolife, Västra Frölunda, Swedish) until the day of transfer, which was performed 48–72 h after oocyte retrieval. Cleaving embryos were evaluated for cell number, blastomere appearance, and fragmentation rate. The best embryos were selected for transfer, and additional high-quality embryos were frozen. Two embryos from each couple were transferred using ultrasound guidance and patients received luteal support, including 600 mg of vaginally administered micronised progesterone (Besins Healthcare, Gabian, Monaco). Serum hCG levels were measured 14 days after embryo transfer and clinical pregnancy was defined as the presence of a visible sac and foetal heartbeat 7 weeks after embryo transfer.