Abstract
Study question
Can whole exome sequencing (WES) of spermatozoal DNA provide insight into understanding the different steps that lead to inability of a couple to reproduce?
Summary answer
The identification of germline mutations can clarify different aspects of reproductive failure in couples with unexplained infertility.
What is known already
The limitation of a routine semen analysis in evaluating sperm characteristics, even according to the most stringent criteria, lies in its inability to provide substantial information on spermatozoa performance in ART. As a result, ancillary tests are being used to further assess the male gamete’s reproductive potential. More recently, WES of the male genome, carried out on somatic cells, has become a powerful technique that can potentially shed light on the genetic causes of infertility. Here, we aim to preferentially detect germline mutations by sequencing spermatozoal DNA to pinpoint genes related to different underlying etiologies for reproductive failure.
Study design, size, duration
In a 5-year period, 25 couples subdivided according to their ICSI outcomes were included in this study. Sperm aneuploidy assessment by fluorescent in situ hybridization (FISH) and copy number variant (CNV) analysis by WES were carried out on ejaculated specimens from consenting male partners. Following CNV analysis, gene mutation profiles were compared between the fertile (n = 10) and infertile cohorts (n = 15), as well as in relation to the reasons for reproductive failure.
Participants/materials, setting, methods
FISH was performed on at least 1,000 sperm cells with a threshold of > 1.6%. DNA was extracted and amplified from at least 500 spermatozoa (DNA concentration, 605±137 ng/ul; quality, 1.7±0.1 nm) for CNV analysis by WES. Mutations corresponding to the CNV were annotated and assessed using the CLC Genomic Server 9.0. Genes were considered duplicated or deleted when their read depth was >1.5 or < 0.5 times the median read depth in the control.
Main results and the role of chance
Couples (n = 25) (maternal age, 38.6±3yrs; paternal age, 39.7±5yrs) had normal somatic karyotypes with normal semen parameters (59.2±30x106/mL concentration, 44.8±18% motility) by WHO standards.
The fertile (n = 10) cohort underwent 12 ICSI cycles, achieving an 82.6% (57/69) fertilization rate and 10/12 (83.3%) term pregnancies. The infertile cohort (n = 15) underwent 21 ICSI cycles, achieving a 66% (62/84) fertilization rate and 5/17 (29.4%) clinical pregnancies, all resulting in pregnancy loss.
Sperm aneuploidy was consistently higher in the infertile (8.4%) versus fertile (4.0%) cohort (P<0.00001), as observed by FISH and DNA sequencing.
For both cohorts, WES detected deletions responsible for sperm–egg fusion (ADAM3A) and acrosomal development (SPACA, SPATA), explaining the necessity for ICSI in these couples.
The infertile cohort was characterized by 4 reasons for cycle failure: complete fertilization failure, poor embryo development, implantation failure, and pregnancy loss. Couples with complete fertilization failure (n = 4) had deletions (PLCZ1, PIWIL1) indicating a sperm-related oocyte-activating deficiency. Those with poor embryo development (n = 5) had mutations (HAUS1, KIF4A, XRN1) essential for centrosome integrity and spindle/microtubular stabilization. Couples who did not achieve pregnancy (n = 7) had a mutation (IL9R) in common related to cytokine constituents in the implantation pathway. Those with pregnancy losses (n = 5) had mutations (NLRP7, TP53) on post-implantation genes.
Limitations, reasons for caution
Several germline mutations, related to the different reasons for these couples’ reproductive failure, were identified. Although intriguing, these findings are still new and need to be validated in a larger study population. In addition, while maternal age was controlled for, we cannot definitively exclude other confounding female factors.
Wider implications of the findings
Additional screening methods for infertile couples, particularly those with unexplained infertility, can be used to clarify elusive factors underlying their reproductive ability. A genetic screening of spermatozoal DNA may therefore be considered a potential tool in precision medicine for the treatment of subtle male factor infertility.
Trial registration number
n/a
Novel WEE2 compound heterozygous mutations identified in patients with fertilization failure or poor fertilization