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New Insights into Male Infertility and Sperm Biology
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New Insights into Male Infertility and Sperm Biology

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biology".

Deadline for manuscript submissions: 20 January 2026 | Viewed by 5992

Special Issue Editor

Dr. Monica Muratori

E-Mail Website
Guest Editor
Department of Experimental and Clinical Medicine, University of Florence, Viale Pieraccini, 6, 50139 Florence, Italy
Interests: male infertility; sperm biology; oxidative stress; sperm DNA fragmentation; semen analysis; flow cytometry; nutrition; environmental pollution
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleague,

Male infertility affects about 7% of men, and, in a large percentage of patients, the causes are unknown. This scenario is expected to worsen in future years as recent data have reported a heavy and still-occurring worldwide decline in sperm count, likely due the deep changes in lifestyle and environmental factors. Routine semen analysis is the cornerstone test for diagnoses of male infertility, but subtler sperm defects than those detected through this test are likely involved. Assisted reproductive technologies provide a chance at parenthood for infertile couples, but their success rate is limited due to the iatrogenic damage occurring during the in vitro manipulation of gametes and embryos.

Hence, there is a need to continue to investigate both the causes of infertility and of damage in sperm structure and function, as well as the factors associated with the decline in the sperm potential of fecundation. Studies concerning sperm DNA fragmentation, oxidative stress, and nuclear condensation have developed our knowledge, but there are still uncertainties to be addressed before they can thoroughly benefit the clinical practice. Furthermore, high-throughput techniques, genomics, epigenomics, transcriptomics, proteomics, and metabolomics are expected to provide new insights into the molecular mechanisms of spermatogenesis and sperm function. The goal is to find new strategies of prevention and treatments for male infertility and for sperm populations used in ART.

Being led by Dr. Monica Muratori and assisted by our Topical Advisory Panel Member Dr. Oumaima Ammar (University of Florence), this Special Issue will focus on novel insights concerning sperm markers for male infertility that have been established by recent research or are promising according to emerging data. With your distinguished expertise in this area, we invite you to consider submitting a contribution to this Special Issue.

Dr. Monica Muratori
Guest Editor

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Keywords

  • male infertility
  • sperm DNA fragmentation
  • sperm oxidative stress
  • sperm nuclear condensation
  • assisted reproductive technologies
  • genomics
  • epigenomics
  • transcriptomics
  • proteomics
  • metabolomics

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Published Papers (4 papers)

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Research

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13 pages, 3951 KB  
Article
Development and Optimization of an RNA-Isolating Protocol for Mammalian Spermatozoa
by El Oulidi Mounia, Azzouzi Naoual, Ravel Celia, Hassani Idrissi Hind, Habbane Mouna, Fieni Francis, Galibert Francis and Akhouayri Omar
Int. J. Mol. Sci. 2025, 26(22), 11171; https://doi.org/10.3390/ijms262211171 - 19 Nov 2025
Viewed by 418
Abstract
The RNAs present in spermatozoa play a crucial role in reproduction and embryonic development. They represent a promising diagnostic tool for assessing male infertility. However, their extraction is challenging due to their low concentration and highly condensed chromatin structure, as well as the [...] Read more.
The RNAs present in spermatozoa play a crucial role in reproduction and embryonic development. They represent a promising diagnostic tool for assessing male infertility. However, their extraction is challenging due to their low concentration and highly condensed chromatin structure, as well as the presence of numerous cellular contaminants. These challenges vary across species and require the development of an optimized and reliable isolation method to obtain high-quality RNAs, which is essential for further molecular analyses regarding the roles played by these RNAs. This study evaluated two RNA extraction methods for spermatozoa in humans and other mammals (dogs, stallions, and bulls): a standard method using the NucleoSpin RNA® II kit (Macherey-Nagel) and an optimized method that combined this kit with dithiothreitol and TRIzol™ pretreatment. In addition, the samples underwent pre-purification to eliminate somatic cells. The optimized method produced a significantly higher total RNA yield along with better purity, which was confirmed by the absence of the 18S and 28S ribosomal RNA peaks, indicating the absence of somatic cell contamination. Additionally, RT-PCR was performed to validate the presence of sperm-specific markers. The quality of the extracted RNAs was assessed by sequencing the mRNA encoding the human olfactory receptor OR1D2 and observing the resulting band on an agarose slab gel with a size corresponding to its entire open reading frame. By addressing long-standing challenges in sperm RNA isolation, our method provides an easy and standardized technique for research in reproductive biology and for advancing our understanding of paternal contributions to transgenerational inheritance. Full article
(This article belongs to the Special Issue New Insights into Male Infertility and Sperm Biology)
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18 pages, 1619 KB  
Article
Induction of Sperm DNA Fragmentation by Cryopreservation and In Vitro Incubation: Comparison of TUNEL, SCSA, SCD Test and COMET Assay
by Costanza Calamai, Michele Tanturli, Donata Conti, Giorgio Leter, Linda Vignozzi, Lisa Giovannelli and Monica Muratori
Int. J. Mol. Sci. 2025, 26(18), 8978; https://doi.org/10.3390/ijms26188978 - 15 Sep 2025
Viewed by 2492
Abstract
TUNEL, SCSA, SCD test and COMET assay are the current tests for detection of sperm DNA fragmentation (sDF) in clinical practice. These four tests are very different from each other for many aspects, possibly including the type of revealed damage. To verify how [...] Read more.
TUNEL, SCSA, SCD test and COMET assay are the current tests for detection of sperm DNA fragmentation (sDF) in clinical practice. These four tests are very different from each other for many aspects, possibly including the type of revealed damage. To verify how the same type of damage was revealed, we simultaneously detected sDF by the four tests before and after induction of sperm DNA breakage by cryopreservation and in vitro incubation. We found that all tests revealed the increase in sDF in both experimental conditions. However, when we pairwise compared the fold increases in induced sDF, we found poor (i.e., values below 0.5) Lin’s concordance correlation coefficients (CCCs) both during cryopreservation and in vitro incubation. The only exception was for SCD test/COMET assay where the CCCs were about 0.5 (cryopreservation: 0.456 (95% CI −0.071–0.784) and incubation: 0.523 (95% CI −0.018–0.827)). Bland–Altman plot analysis showed that TUNEL reveals the highest amounts of sDF during cryopreservation, whereas LiveTUNEL indicated that such damage is undergone by the viable sperm fraction. This is the first study comparing the four tests in detection of sperm DNA damage during cryopreservation and incubation. Full article
(This article belongs to the Special Issue New Insights into Male Infertility and Sperm Biology)
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19 pages, 5669 KB  
Article
Hedgehog Signaling Functions in Spermatogenesis and Keeping Hemolymph–Testis Barrier Stability in Eriocheir sinensis
by Jun-Jie Yu, Hong-Yu Qi, Zhan Zhao, Yu Yang, Shuang-Yi Zhang, Fu-Qing Tan and Wan-Xi Yang
Int. J. Mol. Sci. 2025, 26(11), 5378; https://doi.org/10.3390/ijms26115378 - 4 Jun 2025
Cited by 2 | Viewed by 1234
Abstract
Hedgehog (HH) signaling plays important roles in the development of the nervous system (Sonic hedgehog), bone, cartilage (Indian Hedgehog) and testis (Desert Hedgehog). Research on HH and testes has mostly been conducted in HH-knockout mice and rats, etc. The relationship between HH [...] Read more.
Hedgehog (HH) signaling plays important roles in the development of the nervous system (Sonic hedgehog), bone, cartilage (Indian Hedgehog) and testis (Desert Hedgehog). Research on HH and testes has mostly been conducted in HH-knockout mice and rats, etc. The relationship between HH and cellular junctions has mostly been found in the nervous system and intestine. However, few research studies concerning the link between HH signaling and cell junctions in testis function have been reported. We identified the members of HH signaling that are involved in Eriocheir sinensis testes: HH, Smoothen, Patched, Kif27 and Ci. HH has only one homolog in E. sinensis and is expressed in several types of germ cells in the testes. We found that Kif27 colocalized with Ci in the testes. The knockdown of HH induced enlarged interstitial spaces of the seminiferous tubules. A biotin–streptavidin immunofluorescence experiment indicated that the hemolymph–testis barrier (HTB) was disrupted. Western blot results showed that pinin, HH signaling and cell proliferation- and apoptosis-related protein levels were downregulated. Further immunofluorescent results showed the dislocation of several junction proteins, the abnormality of F-actin and the slowdown of germ cell proliferation and apoptosis. While β-catenin entered the spermatocyte nucleus, it did not activate Wnt-β-catenin signaling, which indicated that the disturbance of the cell cycle in germ cells was not caused by Wnt-β-catenin signaling. In summary, HH signaling plays some roles beyond our understanding in the regulation of the HTB and the germ cell cycle in E. sinensis testes. Full article
(This article belongs to the Special Issue New Insights into Male Infertility and Sperm Biology)
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Review

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20 pages, 1256 KB  
Review
Hurdles of Sperm Success: Exploring the Role of DNases
by Jaime Gosálvez, Carmen López-Fernández, Javier Bartolomé-Nebreda and Carlos García de la Vega
Int. J. Mol. Sci. 2025, 26(14), 6789; https://doi.org/10.3390/ijms26146789 - 15 Jul 2025
Cited by 1 | Viewed by 1169
Abstract
The incidence of sperm DNA fragmentation (SDF) in the ejaculate has garnered increasing attention in recent years due to its negative impact on reproductive outcomes. SDF involves two primary types of damage to the canonical double helix of DNA: single-strand breaks and double-strand [...] Read more.
The incidence of sperm DNA fragmentation (SDF) in the ejaculate has garnered increasing attention in recent years due to its negative impact on reproductive outcomes. SDF involves two primary types of damage to the canonical double helix of DNA: single-strand breaks and double-strand breaks. Both of these can occur throughout the entire process of gametogenesis. Determining the precise causes of elevated SDF remains challenging, as it is influenced by a wide range of physiological processes and environmental factors. This review comprehensively explores the mechanisms underlying SDF, with a particular emphasis on the critical role of deoxyribonucleases (DNases) across different stages of male gamete development, as well as their relevance in assisted reproductive technologies (ART). Full article
(This article belongs to the Special Issue New Insights into Male Infertility and Sperm Biology)
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