Aneuploidy in sperm of fertile men and patients with impaired fertility

The review presents generalized current data on sperm aneuploidy in healthy (fertile) men and infertile male patients with a normal karyotype and with chromosomal abnormalities. The mechanisms of aneuploidy in germ cells, factors affecting of its level, the relationship with defects of spermatogenesis, meiosis, decreased sperm parameters, as well as the effect of sperm aneuploidy on male fertility, embryo development and gestation are discussed.

Male fertility: a review of the publications from April – June 2021

The article provides an overview of the most significant publications on the topic of male infertility. The main selection criteria were considered the practical significance of the article, as well as the impact factor of the journal in which it was published, according to the SCImago Journal Rank (SJR). As a result, a list of 10 works published in the II quarter (April – June) of 2021 was formed. The review includes articles on the following issues: the effectiveness of repeated micro-TESE in non-obstructive azoospermia, the role of COVID-19 in male fertility, the effect of testosterone therapy on spermatogenesis, testicular microlithiasis, electroejaculation as a method of obtaining spermatozoa, harm from carrying cell phones near the genitals, prediction of the effectiveness of intrauterine insemination, the effect of advanced paternal age on sperm aneuploidy, and the importance of the microbiome for male fertility.

FISH and Chimps: Insights into Frequency and Distribution of Sperm Aneuploidy in Chimpanzees (Pan troglodytes)

Numerical chromosomal aberrations in sperm are considered to be a major factor in infertility, early pregnancy loss and syndromes with developmental and cognitive disabilities in mammals, including primates. Despite numerous studies in human and farm animals, the incidence and importance of sperm aneuploidies in non-human primate remains mostly undetermined. Here we investigated the incidence and distribution of sperm aneuploidy in chimpanzees (Pan troglodytes), the species closest to human. We identify evolutionary conserved DNA sequences in human and chimpanzee and selected homologous sub-telomeric regions for all chromosomes to build custom probes and perform sperm-FISH analysis on more than 10,000 sperm nuclei per chromosome. Chimpanzee mean autosomal disomy rate was 0.057 ± 0.02%, gonosomes disomy rate was 0.198% and the total disomy rate was 1.497%. The proportion of X or Y gametes was respectively 49.94% and 50.06% for a ratio of 1.002 and diploidy rate was 0.053%. Our data provide for the first time an overview of aneuploidy in non-human primate sperm and shed new insights into the issues of aneuploidy origins and mechanisms.

P–042 Impact of semen parameters, sperm DNA fragmentation and sperm aneuploidy in male infertility

Study question Should sperm aneuploidies and sperm DNA fragmentation (sDNAfrag) be included as valid tests in the routine investigation of male infertility? Summary answer Sperm DNA fragmentation was associated with male age, oligozoospermia (OZ), oligoteratozoospermia (OT), astenoteratozoospermia (AT) and oligoastenoteratozoospermia (OAT). Sperm aneuploidies were associated with OT and OAT. What is known already Semen parameters assist male infertility diagnosis and treatment, but sDNAfrag and aneuploidy analysis could add useful information, as abnormal values compromise fertility. To include these tests in the routine diagnosis it should be determined if behave as informative parameter and add information regarding the fertility status. For that, further studies comparing these tests to semen parameters are needed, since previous results are not consensual. Additionally, standardization of a sDNAfrag cut-off is needed, as different sample sizes and techniques originate distinct results. Also, until a standardization of the protocol is missing, a cut-off value should be defined for each laboratory. Study design, size, duration A retrospective and prospective investigation was performed, within a 12 years period (April 2007-December 2019). A total of 835 infertile males with a normal karyotype (46,XY) were included. Karyotyping and evaluation of sDNAfrag and sperm aneuploidies were made at a public Genetic unit. All normozoospermic (NZ) patients with a born child and patients whose infertility treatments were done due to female factors were selected from our database and used as controls (60 individuals). Participants/materials, setting, methods Semen analysis followed WHO–2010 guidelines. sDNAfrag was evaluated using the TUNEL assay. Sperm aneuploidies were detected using FISH (chromosomes 13, 18, 21, X, Y). Several tests were applied: correlations for linear associations between numerical variables, ANOVA for comparisons between means, Dunn-test for post-hoc comparisons. To determine the sDNAfrag cut-off value, the area under the ROC curve, sensitivity and specificity, were calculated, with the Youden-Index used to find a threshold that maximizes both sensitivity and specificity. Main results and the role of chance Regarding male age, it was observed a positive correlation with sperm concentration, a negative correlation with sperm vitality (VT) and hypoosmolality, and a positive correlation with sDNAfrag. Regarding sDNAfrag, it was observed negative correlation with sperm concentration, total progressive motility (TPM), morphology, VT and hypoosmolality. Regarding sperm aneuploidies, both total sperm aneuploidy and total sperm disomy exhibited a negative association with sperm concentration, TPM and morphology. It was also investigated whose groups of individuals could be indicated for sDNAfrag or sperm aneuploidy testing. The NZ group evidenced significant lower sDNAfrag, total sperm aneuploidy and total sperm disomy in relation to the non-NZ group. In the NZ group, sDNAfrag was significantly lower in relation to the OZ, OT, AT and OAT groups. The NZ group presented significant lower percentages of sperm aneuploidy in relation to the OT and OAT groups, and significant lower percentages of sperm disomy in relation to the OAT group. Additionally, sDNAfrag was positively correlated with total sperm aneuploidy and total sperm disomy. From the present large population, ROC curve analysis allowed estimating a cut-off value of 18.8% for the TUNEL-assay (sDNAfrag), with 0.658 of area under the curve, 53.9% sensitivity and 76.7% specificity. Limitations, reasons for caution Although presenting a high number of cases and strict controls, the present study was unable to include as controls healthy men with proven fertility. Additionally, the present study did not take into account life-style factors and male associated pathologies besides infertility. Wider implications of the findings: Semen parameters were shown to be negatively correlated with sDNAfrag and sperm aneuploidies. As sDNAfrag testing and sperm aneuploidy testing were associated with semen abnormalities and male age, it is suggested their inclusion in the routine evaluation of infertile men, thus adding important complementary information about the fertility status. Trial registration number Not Appliable

P–057 Preliminary evaluation of laser confocal Raman spectroscopy as a noninvasive method for detecting sperm chromosome aneuploidy

Study question To evaluate the efficiency and accuracy of Raman microspectra in detecting sperm chromosome balance state by DNA content difference. Summary answer Raman spectroscopy can identify the difference of X and Y sperm DNA content, but the accuracy still needed to be improved for clinical application. What is known already Aneuploid sperm fertilization affects embryo quality and leads to the waste of oocytes in Assisted Reproductive Technology (ART). Raman spectroscopy can identify substances and observe molecular changes through specific spectral patterns with high specificity and has become a new hot spot in ART. Previous research has used this technology to detect embryo culture medium to evaluate the aneuploidy of embryos. The DNA content of X and Y in sperm was different, which may serve as a marker for sperm aneuploidy detection by Raman spectroscopy. Study design, size, duration The significant difference in the morphology of the sex chromosomes of X and Y spermatozoa leads to a substantial difference in the DNA content. We perform Raman spectroscopy to identify the spectral differences of the sperms, especially the differences in sperm DNA content. We further verified the accuracy with fluorescence in situ hybridization (FISH). Participants/materials, setting, methods Spermatozoa were provided by healthy donors with normal aneuploidy, and analysis parameters met the current World Health Organization (WHO, 2010) standards. Sperm heads were detected by laser confocal Raman spectroscopy and obtained the corresponding spectra. The sperm chromosome information was classified by Standard principal component analysis (PCA) and identified by fluorescence in situ hybridization (FISH). Student’s t-test and Receiver operating characteristic (ROC) curve analysis was performed for further analysis. Main results and the role of chance Standard principal component analysis (PCA) after unqualified quality control divided spermatozoa into two groups according to the calculation and calibration results, 22 cases in group A and 31 cases in group B. Then, we conducted frequency distribution histogram statistics on the above data, and the results showed that there were differences in frequency distribution at I785 = 23,750 and Area714 –1162 = 3,250,000. The FISH analysis identified sex chromosomes of 59 spermatozoa, which was not exactly one-to-one correspondence with the results of PCA analysis. Then we further analyzed the sperm of 59 cases by statistical analysis. The results showed that there were significant differences between X sperm (n = 39) and Y sperm (n = 20) at 714–1162 cm–1 and 785 (P < 0.05). ROC curve analysis was used to evaluate the sensitivity of correlation between sperm DNA content and Raman spectra. The results showed that the corresponding thresholds of I785 = 24,986.5 and Area714–1162 cm–1 = 3,748,990 were the best for distinguishing the two kinds of sperm. When the sperm’s peak value of 785 or 714–1162cm–1 exceeds the above thresholds, X-sperm’s possibility greatly increased. The AUC of the ROC curve in both cases was 0.662 and 0.696, respectively. Limitations, reasons for caution Current Raman spectroscopy requires spermatozoa elution and fixation, which damage the sperms. Furthermore, current Raman spectral data are not obtained from the whole sperm head, limiting the accuracy of this technique. Wider implications of the findings: Our results indicated that Raman spectroscopy had potential application value for sperm aneuploidy detection and could be used as a noninvasive selector for normal haploid sperms in the ART. Trial registration number LL-SC–2018–038

O-120 Unravelling reproductive failure by scrutinizing the male germline code

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

Single-center thorough evaluation and targeted treatment of globozoospermic men

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.

Analysis of sperm chromosomes in six carriers of rare and common Robertsonian translocations

Introduction: Robertsonian translocation (RobT) is the central fusion of the long arms of two acrocentric chromosomes, leading to 45 chromosomes in humans. The most common ones are rob(13;14) and rob(14;21) (91%). Other types of RobT are so-called rare cases. In the general population RobTs occur with a frequency of approximately 0.123%, but among men with reproductive failure this value rises 9-fold. Infertility in RobT carriers is associated with the formation of unbalanced spermatozoa resulting from segregation of the chromosomes involved in trivalent during the meiotic prophase. In spermatozoa of many RobT carriers an increased level of chromosomal aneuploidy is observed. Materials and Methods: We examined the hyperhaploidy level of chromosomes 7, 9, 18, 21, 22, X and Y in spermatozoa of 6 RobT unrelated carriers: two carriers with rare rob(13;15), one with rare rob(13;22), and three of the common rob(13;14). Results were compared with the control data from a group of 7 fertile men with a normal karyotype. Fluorescent in situ hybridization (FISH) was applied. Results: We found an increased level of sperm aneuploidy regarding at least one of the analyzed chromosomes in each of the carriers, while in rare RobTs interchromosomal effect (ICE) was observed. Meiotic segregation pattern of a rare rob(13;15) carrier revealed the 76% of normal /balanced spermatozoa. Disucussion: Due to the relatively high population frequency of RobTs, their influence on reproductive failure, hight risk of imbalancement in prenatal diagnosis (7%), and small amount of data for rare RobTs, each newly characterized case is valuable in genetic counseling.

Frequency of Sperm Aneuploidy in Oligoasthenoteratozoospermic (OAT) Patients by Comprehensive Chromosome Screening: A Proof of Concept

Background: Embryonic aneuploidy usually results in implantation failure and miscarriage. Considering significantly high frequency of sperm aneuploidy reported in oligoasthenoteratozoospermia (OAT) using fluorescence in situ hybridization (FISH) in limited number of chromosomes and lack of comprehensive chromosome screening (CCS) in OAT, the aim of this study was applying CCS in OAT sperm and comparison of the results with FISH findings. Methods: Five OAT patients with normal blood karyotypes and history of implantation failure were included. The successfully amplified samples, each containing two sperm, were analyzed by array comparative genomic hybridization (aCGH). FISH was utilized mainly depending on the aneuploidies found by aCGH to assess their frequencies in total sperm population. Results: In aCGH for 30 sperm, aneuploidy was found in 66% of samples. Following the study of 4300 sperm by FISH, an average of 55.46% aneuploidy was observed. No pregnancy was resulted with normal partners. Conclusion: Using aCGH, some abnormalities were observed that are not typically considered in sperm FISH studies. Despite small sample size of the comprehensive study, like other similar studies, the frequency of aneuploidies was considerable and similar to FISH. Aneuploidies revealed by aCGH at single sperm resolution were different from sperm population detected by FISH. Considering high frequency of aneuploidy in OATs sperm, preimplantation genetic testing for aneuploidy (PGT-A) can be used for in transfer of chromosomally normal embryos.