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Objective To determine if 6-month folic acid (5 mg) and zinc (30 mg) supplementation impacts sperm DNA methylation patterns. Design A multicenter, double-blind, block randomized, placebo-controlled trial titled “The Folic Acid and Zinc Supplementation Trial (FAZST).” Setting Infertility care centers. Patient(s) Male partners (18 years and older) from heterosexual couples (female partners aged 18–45 years) seeking fertility treatment were recruited. Intervention(s) Men were randomized 1:1 to receive folic acid (5 mg) and elemental zinc (30 mg) (n = 713) or a matching placebo (n = 757) daily for 6 months. Main Outcome Measure(s) Sperm DNA methylation was analyzed using the EPIC methylation array (Illumina) at 6 months. Differential sperm DNA methylation was assessed at multiple levels (regional, single cytosine phosphate guanine, etc.). We additionally assessed the impact of supplementation on epigenetic age. Result(s) No significant differences were identified between the treatment and placebo groups although some trends appeared to be present. To determine if these trends were noteworthy, we implemented various permutations and found that the patterns we identified were no more than would be expected by random chance. Conclusion(s) The data presented here strongly suggest that this supplementation regimen is not effective at altering sperm DNA methylation. These data comport well with previous findings from the FAZST study that found no impact of supplementation on basic semen analysis parameters or live birth. Clinical Trial Registration Number ClinicalTrials.gov Identifier: NCT01857310.

Objective To determine the precision and accuracy of an automated cell counting system when applied to counting aneuploidies in sperm samples. Design Prospective pilot study. Setting Andrology clinic and research laboratory in a university teaching hospital. Patient(s) Ten anonymous sperm donors of known fertility and two patients seeking infertility treatment. Intervention(s) Semen samples were processed for detection of aneuploidies for chromosomes 13, 18, 21, X, and Y with use of fluorescent in situ hybridization. The detection of chromosome aneuploidy was performed both by manual counting and by the use of an automated cell counting system with manual review of aneuploid sperm. Main Outcome Measure(s) Semen samples were judged for the percent aneuploidy for chromosomes 13, 18, 21, X, and Y when counted manually or with the use of the automated cell counting system and review by a technician. Result(s) The sperm aneuploidy rates determined by the automated cell counting system and careful review were comparable with those obtained by manual counting by a trained technician. Conclusion(s) These preliminary data demonstrate that automated cell counting devices may be useful in increasing productivity in aneuploidy detection in sperm and may become an alternative to the labor-intensive manual counting by technicians.

Objective: To characterize and contrast protamine levels, chromatin decondensation, chromosome aneuploidy rates, and functional ability of round-headed sperm from two brothers with round-headed sperm syndrome. Design: Analysis of semen samples from siblings with round-headed sperm syndrome and comparison with semen of fertile donors. Setting: University school of medicine and laboratories. Patient(s): Two infertile siblings. Main Outcome Measure(s): Sperm aneuploidy rates of chromosomes X, Y, 13, 18, and 21, protamine 1 and 2 (P1-P2) ratios, Western blot evaluation of protamines, and chromatin decondensation rates. Additional measures include standard semen quality parameters, electron microscopy ultrastructure evaluation, analysis of acrosome morphology, and sperm penetration rates. Result(s): Aneuploidy rates were significantly increased in sibling no. 1 but not in sibling no. 2. The levels of P1 and P2 were decreased in sibling no. 1, and an unusual level of protamine precursors was present. Ultrastructural differences also were observed between the siblings. Conclusion(s): These data indicate profound differences in sperm from two siblings with complete round-headed sperm syndrome. Multigenic defects and/or variable expression of the syndrome may be responsible for the syndrome and necessitate individual screening of affected individuals because the pattern of expression appears highly variable.

Objective To evaluate sperm ultrastructure and the efficacy of intracytoplasmic sperm injection (ICSI) with careful positioning of the sperm midpiece next to the sperm head in a patient with easily decapitated sperm syndrome. Design In vitro fertilization case report with retrospective sperm ultrastructure analysis. Setting In vitro fertilization clinic and andrology laboratory. Patient(s) A couple seeking treatment for diminished ovarian reserve and male factor infertility using donor oocyte IVF. Intervention(s) Motile sperm inadvertently decapitated during micromanipulation were used for a modified ICSI procedure in which the midpiece was carefully positioned proximal to the sperm head. Sperm were also analyzed by transmission electron microscopy (TEM). Main outcome measure(s) Fertilization rate, embryo development, pregnancy, and the incidence of normal sperm ultrastructure. Result(s) The ICSI resulted in a fertilization rate of 63% and embryo development of good to moderate quality of 36% of the embryos. Transfer of two embryos resulted in a pregnancy and birth of a healthy child. Normal sperm centrosomes were identified retrospectively. Conclusion(s) Pregnancy can result in patients where the sperm are decapitated during immobilization before ICSI if normal centrosomes are present and the head and midpiece are positioned closely together.

Objective To determine the origin of large plaque-like cellular and acellular masses of debris found in the semen of a patient with otherwise normal semen quality and secondary infertility. Design A prospective case study. Setting Academic andrology laboratory. Patient(s) Secondary infertility patient presenting for routine semen evaluation. Intervention(s) Following diagnosis of a seminal infection, the patient was treated with an antibiotic. Main outcome measure(s) Semen quality parameters, semen culture, and electron microscopy identification of cytolysosomal spermophagy. Result(s) Semen culture identified a rare infection with Ralstonia picketti and electron microscopy showed massive neutrophil and cytolysosomal spermophagy. Both were eliminated after antibiotic therapy. Conclusion(s) This case study documents a rare infection of the reproductive tract with Ralstonia picketti . Analysis of large seminal masses determined their origin to be primarily from acellular cytolysosomes. The presence of such masses in semen should be further evaluated, including semen culture.

Objective To investigate YBX2 gene alterations in men with severe defects in spermatogenesis, including azoospermia or severe oligozoospermia, and protamine deregulation. MSY2 has been identified as a central component in the regulation of spermatogenesis in mice, but the potential role of its human orthologue, YBX2 or "Contrin," in human infertility is not known. Design A prospective cohort study. Setting University infertility clinic and associated research laboratory. Patient(s) A total of 288 men were evaluated. Diagnoses were made of complete azoospermia, severe oligozoospermia, and protamine deregulation, or men were of known paternity. Intervention(s) Deoxyribonucleic acid (from peripheral blood) and semen samples were collected and analyzed for gene mutations and semen parameters respectively. Main Outcome Measure(s) YBX2 gene alterations. Result(s) YBX2 sequence analysis revealed 15 polymorphic sites, of which seven polymorphisms were present at a statistically higher frequency in one or both of the patient populations than in controls. Of these seven, two resulted in an amino acid substitution in the highly conserved cold shock domain and one resulted in a highly significant synonymous change in exon 8 of infertile patients. The frequency of single nucleotide polymorphisms was significantly elevated in patients with infertility, particularly in men with abnormal protamine expression. Conclusion(s) These data indicate a significant association between gene alterations in the YBX2 gene and abnormal spermatogenesis in humans, including a potential role in altering protamine expression, and implicate YBX2 gene alterations as a potential cause of male factor infertility.

Objective To develop a technique with the potential of isolating genetically fit sperm for assisted reproductive technology (ART) treatment without compromising its structural or functional competence. Design Observational study. Setting University hospital. Patient(s) Fifty patients undergoing infertility diagnosis and 88 couples undergoing ART treatment. Intervention(s) None. Main Outcome Measure(s) Under an electric field, the percentage of positively charged sperm (PCS), negatively charged sperm (NCS), and neutrally charged sperm was determined for each ejaculate before and after density gradient centrifugation (DGC), and evaluated for sperm DNA damage, histone retention, and couples' ART outcomes. Subsequently, PCS, NCS, and neutrally charged sperm were selected using an intracytoplasmic sperm injection needle and directly analyzed for DNA damage. Result(s) There was a reduction in the NCS population (95.10% ± 0.94% vs. 54.48% ± 2.39%) and an increase in the PCS population (4.28% ± 0.58% vs. 42.52% ± 2.36%) after DGC. The DNA damage was inversely proportional to %NCS ( r 2 = −0.242) and directly proportional to the %PCS ( r 2 = 0.206). When sperm were picked according to their charge and directly analyzed, sperm DNA damage was lower in the NCS population (3.9% ± 1.5%) compared with control (17.3% ± 3.2%) and %PCS populations (27.8% ± 6.0%). The %NCS was positively associated with fertilization rate ( r 2 = 0.469) and blastocyst development ( r 2 = 0.308) and inversely associated with embryo arrest ( r 2 = −0.253). Implantation rate and clinical pregnancies were higher in patient groups with increased NCS. Conclusion(s) Selection of NCS through micro-electrophoresis has the potential to isolate sperm relatively free of DNA damage to be used in ART.

Sperm protamine-1 (P1) and protamine-2 (P2) concentrations were evaluated concomitantly with global DNA methylation patterns in a population of male infertility patients. Protamine quantification and immunofluorescence microscopy in conjunction with quantitative image analysis revealed no significant relationships between the P1/P2 ratio, P1 concentrations, or P2 concentrations and levels of global DNA methylation in mature spermatozoa.

Failed fertilization and the appearance of immature oocytes are common in IVF practice; rescue intracytoplasmic sperm injection can be used as a therapy. However, this study indicates that embryos created after in vitro maturation and delayed intracytoplasmic sperm injection contain an increase in aneuploidy (79.7%) over control embryos (60.5%). Therefore, patients should be informed of the possible risk when presented with delayed intracytoplasmic sperm injection.