Purpose To characterize, by specific biomarkers and nucleic acid sequencing, the structural and genomic sperm characteristics of partial (PG) and complete globozoospermic (CG) men in order to identify the best reproductive treatment. Methods We assessed spermatozoa from 14 consenting men ultrastructurally, as well as for histone content, sperm chromatin integrity, and sperm aneuploidy. Additional genomic, transcriptomic, and proteomic evaluations were carried out to further characterize the CG cohort. The presence of oocyte-activating sperm cytosolic factor (OASCF) was measured by a phospholipase C zeta (PLCζ) immunofluorescence assay. Couples were treated in subsequent cycles either by conventional ICSI or by ICSI with assisted gamete treatment (AGT) using calcium ionophore (Ionomycin, 19657, Sigma-Aldrich, Saint Louis, MO, USA). Results Ultrastructural assessment confirmed complete acrosome deficiency in all spermatozoa from CG men. Histone content, sperm chromatin integrity, and sperm aneuploidy did not differ significantly between the PG (n = 4) and CG (n = 10) cohorts. PLCζ assessment indicated a positive presence of OASCF in 4 PG couples, who underwent subsequent ICSI cycles that yielded a 36.1% (43/119) fertilization with a 50% (2/4) clinical pregnancy and delivery rate. PLCζ assessment failed to detect OASCF for 8 CG patients who underwent 9 subsequent ICSI cycles with AGT, yielding a remarkable improvement of fertilization (39/97; 40.2%) (P = 0.00001). Embryo implantation (6/21; 28.6%) and clinical pregnancies (5/7; 71.4%) were also enhanced, resulting in 4 deliveries. Gene mutations (DPY19L2, SPATA16, PICK1) were identified in spermatozoa from CG patients. Additionally, CG patients unable to sustain a term pregnancy had gene mutations involved in zygote development (NLRP5) and postnatal development (BSX). CG patients who successfully sustained a pregnancy had a mutation (PIWIL1) related to sperm phenotype. PLCZ1 was both mutated and underexpressed in these CG patients, regardless of reproductive outcome. Conclusions Sperm bioassays and genomic studies can be used to characterize this gamete’s capacity to support embryonic development and to tailor treatments maximizing reproductive outcome.
Study question What are the best methods of selecting spermatozoa with the highest genomic integrity in order to improve embryo implantation and term pregnancy rates with ICSI? Summary answer Testicular or ejaculate spermatozoa isolated by microfluidic sperm selection (MFSS) were characterized by superior genomic integrity with improved clinical pregnancy and delivery rates. What is known already In couples with unexplained infertility, a subtle male factor can often be identified. Both single-strand (ss) and double-strand (ds) DNA nicks and breaks hinder the ability of the male gamete to support embryonic development. Surgical retrieval of spermatozoa from the proximal male genital tract can prevent their exposure to oxidative stress. Moreover, use of membrane-based microfluidics chips has been shown to allow for selection of the most progressively motile spermatozoa with higher genomic integrity. Study design, size, duration Over the course of 48 months, 86 consenting men presenting with high sperm chromatin fragmentation (SCF) in their ejaculate with prior ART failure underwent a subsequent cycle with specimens retrieved by testicular biopsy or ejaculate processed by MFSS. A concurrent TUNEL assay was performed on samples collected or selected by each method. Sperm specimens of both origins were utilized for ICSI cycles. Semen parameters, chromatin integrity, and pregnancy outcomes were compared between the two methods. Participants/materials, setting, methods Fresh ejaculates from consenting men were collected for standard semen analysis (WHO 2010). Testicular biopsy and MFSS were used to isolate spermatozoa with a higher genomic integrity after previous ART failure. SCF was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) on at least 500 spermatozoa under a fluorescent microscope with a threshold of ≥ 15%. MFSS was carried out by Zymot® chips. ICSI was performed in the standard fashion. Main results and the role of chance A total of 86 men (36.5±5 years) had the following semen parameters: volume of 2.6 ±1mL, concentration of 27.0±33 x 106/mL, 35.6±15% motility, and high SCF (24.1±10%). They underwent 146 ICSI cycles with their partners (maternal age, 33.7±3) resulting in a high incidence of pregnancy loss (100%; 13/13). Of those who failed to conceive, 22 couples used surgically retrieved spermatozoa (SRS) with a concentration of 1.8 ± 4 x 106/mL (P < 0.01), 5.0±11% motility (P < 0.01), and an SCF of 12.6 ± 6% (P < 0.0001). SRS was used in 37 ICSI cycles, yielding a fertilization rate of 61.6% (204/331, P < 0.01), an implantation rate of 10.6% (9/85, P < 0.01), a CPR of 23.5% (8/34, P < 0.01), and a delivery rate of 17.6% (6/34, P < 0.01). Another 24 couples underwent ICSI cycles with ejaculated spermatozoa processed by MFSS with a concentration of 1.8±3 x 106/mL (P < 0.01), but an increased motility of 99±1% (P < 0.01) and an SCF of 1.2 ±1%, lower than both the raw and testicular specimens (P < 0.0001). MFSS-processed specimens resulted in a fertilization rate of 76% (335/441, P < 0.01), an implantation rate of 26.3% (15/57, P < 0.05), and a CPR of 67.9% (19/28, P < 0.01), of which 15 patients delivered and 2 pregnancies are ongoing (89.5%; P < 0.01). Limitations, reasons for caution This is a preliminary study on a small number of subjects. A randomized prospective study conducted on a larger cohort would be required to confirm our findings. Wider implications of the findings: SCF severely affects pregnancy by impairing embryonic development, consequently promoting implantation failure. While retrieving spermatozoa from the germinal epithelium is a viable option, MFSS provides an alternative. Although MFSS requires an adequate number of sperm with good kinetic characteristics, it provides a more palatable option, reducing surgical risk and costs. Trial registration number Not applicable
Study question Can microfluidic sperm selection (MFSS) select male gametes without sperm chromatin fragmentation (SCF) and double-stranded DNA breaks (dsDNA) in order to generate euploid conceptuses? Summary answer Couples treated by ICSI with MFSS had significantly improved embryo ploidy rates and pregnancy outcomes, demonstrating the efficacy of this novel selection method. What is known already SCF has been linked to infertility, specifically to embryo developmental and implantation failure. This damage can be both single-stranded (ssDNA) or double-stranded (dsDNA). Recent studies have shown that dsDNA in particular causes chromosomal aberrations and contributes to embryo aneuploidy, which leads to implantation failure. Study design, size, duration Consenting couples treated at our center by intracytoplasmic sperm injection (ICSI) with spermatozoa selected by MFSS were included. The majority of these couples had a medical history significant for elevated SCF, recurrent implantation failure, and embryo aneuploidy. ICSI clinical outcome, as well as preimplantation genetic testing for aneuploidy (PGT-A) and frozen embryo transfer (FET), was recorded and compared to the couples’ historical treatments following sperm selection by density gradient centrifugation (DGC). Participants/materials, setting, methods From 2016 to 2020, 51 consenting men had their ejaculates screened for SCF levels by terminal deoxynucleotidyl dUTP transferase nick-end labeling (TUNEL) using a commercially available kit. At least 500 spermatozoa were assessed per patient, with a normal threshold of ≤ 15%. To screen for dsDNA, neutral Comet using a modified in-house protocol was also performed in a pilot study. At least 200 spermatozoa were assessed per patient, with a normal threshold of ≤ 3%. Main results and the role of chance A total of 51 men (average age, 41.0±8 years) had mean sperm concentrations of 39.0±33x106/mL, 38.4±12% motility, and 2.1±1% normal morphology. Following DGC and MFSS, the concentrations were 4.7±8 and 4.3±8x106/mL and the motility was 64.0±33 and 98.0±3%, respectively (P < 0.0001). The average SCF decreased from 20.1±18% in the ejaculate to 16±3% following DGC, but was 2.9±4% after MFSS. The dsDNA fell from 3.4±3% in raw specimens to 2.9±1% after DGC, and to only 0.5±0.7% following MFSS (P < 0.0001). These men underwent ICSI with their female partners (average age, 37.3±4 years), with sperm selected by DGC; they achieved a fertilization rate of 56.4% (337/597) with 26.0% euploid embryos (36/139). FET cycles from these embryos yielded an implantation rate of 8.3% (2/24) and a clinical pregnancy rate (CPR) of 15.4% (2/13), but both miscarried. These couples then underwent ICSI with MFSS, with a fertilization rate of 78.0% (588/754; P < 0.0001) and 50.0% (172/344; P < 0.0001) euploid embryos after PGT-A. A total of 37 embryos have been replaced, with an implantation rate of 67.6% (25/37; P < 0.0001) and a CPR of 73.5% (25/34; P < 0.001), with an ongoing/delivery rate of 70.6% (24/34; P < 0.0001). Limitations, reasons for caution While the oocyte contribution cannot be discounted, MFSS was able to yield spermatozoa that had the highest motility and ability to produce euploid embryos following ICSI. Wider implications of the findings: The genome and epigenome of the spermatozoon, and their contribution to reproductive outcomes, are being vigorously explored and scrutinized. Alternative approaches to gamete selection, such as MFSS, in couples with elevated SCF and dsDNA provide the best chances for future pregnancies by mitigating embryo aneuploidy. Trial registration number Not applicable
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