The Effects of Boron on Sperm Qualities and Testicular Histopathology in Animal Studies: A Systematic Review

Amin Sani; Anis Sani; Soroush Sharifimoghadam; Mahdi Bahari; Masoumeh Emamvirdi; Amir Emamvirdi; Farhad Tondro Anamag; Hanieh Salehi-Pourmehr; Sakineh Hajebrahimi; Mustafa Numan Bucak

doi:10.4274/jus.galenos.2025.2024-9-7

Abstract

This study systematically reviewed how boron exposure affects animal model sperm parameters and testicular structure. The Embase, Scopus, PubMed, ProQuest, and Web of Science databases were searched up to January 2023. The studies that examined boron’s efficacy and safety regarding semen parameters and testicular histopathology in different animal species were included. Studies involving specific derivatives or <em>in vitro</em> or human studies were excluded. Two reviewers selected the studies and extracted the data independently. The quality of eligible studies was assessed using the Animal Research: Reporting of <em>In Vivo</em> Experiments Essential 10 checklist. The outcomes were summarized and presented in tables. Sixteen studies were included from 1,602 retrieved articles. While some studies demonstrated that boron, at doses of 17.5, 35, and 70 mg/kg for 8 weeks, improved the quality of spermatogenesis in terms of rate of sperm movement, sperm concentration, and total volume of sperm, other studies showed dose-dependent boron toxicity to the reproductive system. While some studies suggest potential benefits of boron supplementation on spermatogenesis, others indicate harmful effects. The conflicting results emphasize the need for further research to establish clear guidelines on the appropriate dosage, duration, and safety of boron in improving sperm quality.

Keywords:

Boron, sperm parameters, testicular histopathology, reproductive toxicity, systematic review

Introduction

Boric acid, containing boron, is an inorganic acid often used as a water pH regulator, stabilizer, neutralizer, and buffer in the glass industry, food preparation, beauty industry, agriculture, and pharmaceutical production (1, 2). Obviously, with the development of the industry, the importance of this acid and its amount in our environment has increased due to its widespread use. In recent years, the physiological role of boron has attracted the attention of researchers worldwide, especially in the field of the reproductive system (3). Studies on the effects of boron on animals began in the 1990s. According to the European Union report, boron is placed in group 1B of the Globally Harmonized System classification, specifically in the category R60-61. Group R60 weakens the reproductive system, while group R61 is harmful to the fetus (4).

Although the definite role of boron in the physiology of animals is not completely clear, the effects of its supplementation in the diet on bone growth and in the central nervous, endocrine, and reproductive systems have been reported (5, 6). Boron increases the level of interleukin-6 and regulates serine protease enzyme activity. It also improves the immune and the antioxidant capacity and calcium metabolism (6). In animals, most of the studies on the effects of boron focus on mice and rats. The findings of these studies are interesting because they have produced contradictory results regarding Boron’s activity. The study showed that high concentrations of boron have positive effects on lipid peroxidation reduction and antioxidant metabolism in mice (7). In rabbits, boron improves antioxidant capacity, sperm quality, and testosterone concentration (8). However, high concentrations of boron have negative effects on the reproductive system of animals. Boric acid causes testicular atrophy in rats and dogs at specific doses and concentrations (9). Another study found that a diet with 1,000 ppm boron, reduced the number of spermatocytes, spermatids, and mature spermatozoa in mice after 30 days (10). Additionally, high dietary boron concentrations have detrimental effects on the reproductive system of male rats, and at a certain dose, these concentrations can lead to acute toxicity and death. In animal tissue studies, toxic concentrations of boron damage the process of spermatogenesis, resulting in testicular atrophy in 10-14 days (11).

According to studies on the effect of boron on the reproductive system of animals, it is believed that boron acts as a double-edged sword that can have beneficial or harmful effects on this system, depending on the dose. Therefore, in this study, we aimed to systematically review the effect of boron on sperm parameters and testicular histopathology in animal models.

Methods

This systematic review followed the guidelines of the preferred reporting items for systematic reviews and meta-analyses (PRISMA) Statement and the Cochrane Collaboration Handbook. Due to resource limitations and institutional policies at the time, registration was not feasible; furthermore, the narrowly defined scope of this review was considered sufficient to justify the absence of registration.

Data Sources and Search Strategy

The Embase, Scopus, PubMed, ProQuest, and Web of Science databases were thoroughly searched in January 2023. Finding both published and unpublished studies was the goal of the search approach. A three-step search process was used in this study. Following a preliminary, constrained search of MEDLINE, the title and abstract’s text words were examined. In January 2023, a second search across all included databases was conducted using all indicated keywords and index phrases: ((((“reproduction”[MeSH Terms]) OR (“reproduction”[Title/Abstract])) OR (“reproductive”[Title/Abstract])) OR (((“infertility”[MeSH Terms]) OR (infertil*[Title/Abstract])) OR (Sperm [Title/Abstract]))) AND ((“boron”[MeSH Terms]) OR (boron [Title/Abstract])).

The last step was to look for further studies in the reference lists of all the indicated papers and articles. This review covered studies that were released on any date and in any language.

Inclusion and Exclusion Criteria

The original studies that examined boron’s efficacy and safety of semen parameters and testicular histopathology in different animal species were included. Studies involving specific derivatives or in vitro/human studies were excluded. Also, Abstracts, reviews, letters, and theses were excluded.

Study Selection and Data Extraction

The retrieved articles from multiple information sources were organized using PRISMA flowcharts. Two reviewers independently screened all titles and abstracts of the retrieved articles. Additionally, full texts of relevant studies were independently assessed for eligibility, with reasons for exclusion documented for the full texts that were excluded. Data extraction was performed separately by two researchers, and any discrepancies were discussed and resolved. The following data was extracted from the included studies: Author Name, Publishing Year, Country, Study Design, Animal, Sample Size, Age, Diet, Boron Derivative and Dosage, Intervention Duration, Testis Weight, Testicular Morphometry, Pathology Report, Sperm Count/Concentration, Sperm Abnormality, Sperm Motility, and Ejaculate Volume.

Risk of Bias Assessment

Using the Animal Research: Reporting of In Vivo Experiments (ARRIVE) Essential 10 checklist, two independent reviewers evaluated the methodological quality of the selected articles. The reviewers’ probable differences were settled through conversation or by a third reviewer.

Data Synthesis

The primary outcomes in this study were the change of sperm parameters and testis histopathology in animals with boron exposure. As these outcomes were diverse or heterogeneous (from different animal species), combining the data and conducting a meta-analysis was not possible. The results are summarized and presented in tables.

Results

Study Selection

The electronic search, manual search, and reference check yielded a total of 1,602 citations. After removing duplicate citations, we were left with 919 studies for screening. Based on the titles and abstracts provided, 35 papers were selected, while 19 articles were disqualified during the full-text selection process. Ultimately, 16 studies were included. For more detailed information on the selection procedure, please refer to Figure 1 in the PRISMA flowchart.

Study Characteristics

In this research, 16 studies were reviewed from 1976 to 2022. These studies were conducted in the United States, Japan, Chile, Egypt, Turkiye and India. The investigated animals were rats, mice, rabbits, African clawed frogs, goats, and Osemi Rams.

Results of Individual Studies

As mentioned, the results of the studies were notably different from each other. In the study of Elkomy et al. (12), Ibrahim et al. (11), Krishnan et al. (13), and Abdel-Wahab et al. (14), boron improved the quality of spermatogenesis. In the Elkomy et al. (12) study, after the administration of boron at the doses of 17.5, 35, and 70 mg/kg for 8 weeks, it was seen that the rate of sperm movement, sperm concentration, and total volume of sperm increased compared to the control group (11). Similarly, the study by Ibrahim et al. (11), which gave a 70 mg/kg boron-containing diet to Osmei rams for 4 months, showed an improvement in total sperm volume and sperm concentration (12). Krishnan et al. (13) also administered 40 ppm of this acid in the form of sodium tetraborate to goats for 60 days and concluded that there was a significant increase in sperm motility compared to the control group. Another interesting result was obtained by Abdel-Wahab et al. (14) in 2022 by administering 70 mg/kg of boric acid to goats for 24 weeks; they observed that goats receiving boron exhibited improvements in the structure of testis tubules, and the activity of Sertoli cells increased. Conversely, other studies have shown that boron toxicity affects the reproductive system. Ayranci et al. (15) administered 1000 mg/kg/day of boric acid to Sprague-Dawley rats for 7 days, and observed an increase in edema in the testicular interstitial tissue and an increase in apoptotic cells. El-Dakdoky et al. (16) also gave 125, 250, or 500 mg/kg of boron to Wistar rats for 60 days, which led to the destruction of spermatogonia, spermatocytes, and spermatozoa with a dose of 250 mg/kg and severe destruction of germ cells with a dose of 500 mg/kg. Lee et al. administered boron to Sprague-Dawley rats with doses of 500-1000-2000 ppm for 90 days. They observed that with a dose of 500 ppm, boron did not cause any complications. However, with a dose of 1000 ppm, germ cell destruction began, and the diameter of seminiferous tubules was reduced. Eventually, testicular atrophy occurred (10). Tables 1 and 2 represent the characteristics of the included studies.

Methodological Quality

The risk of bias assessment of included studies is summarized in Table 3. All studies were of good general quality based on the ARRIVE Essential 10 appraisal checklist.

Discussion

Our systematic review showed contrasting findings. While some studies demonstrated that boron improved the quality of spermatogenesis in terms of sperm movement, sperm concentration, and total volume of sperm at the doses of 17.5, 35, and 70 mg/kg, or improvement in the structure of testis tubules and the activity of Sertoli cells, other studies have shown that boron toxicity to the reproductive system is dose-dependent. For example, 1000 mg/kg/day of boric acid increased edema in the testicular interstitial tissue and apoptosis in cells of rats, and it led to the destruction of spermatogonia, spermatocytes, and spermatozoa or germ cells, a reduction of the Seminiferous tubules diameter, and eventually testicular atrophy. Boron is a mineral found in various food sources that has recently received attention for its effect on male fertility (17). Treatment with boric acid in rats, mice, and dogs has been found to reduce fertility and sperm production. However, in some studies, beneficial effects on these processes have been observed, which seem to be dose-dependent. Toxic effects were observed at higher doses, and the substance is beneficial at lower doses. This systematic review examines the studies on boron and its effects on the reproductive system of animals, along with their contradictory results.

According to histological studies, the first toxic effect of boric acid on spermatogenesis occurs in germ cells, after which Sertoli cells are destroyed, and the testicle atrophies (18). The researchers also claimed that these toxic effects begin on the seventh day of administration. Treinen and Chapin (19) showed that this boron toxicity is caused by a decrease in blood testosterone levels. Another mechanism that has been proposed for boron toxicity is its cytotoxic effects on sperm DNA, which increases the DNA’s fragility. Boron also increases oxidative stress in high doses (11). On the other hand, scientists claim that boron in low doses has beneficial effects on the reproductive system. Ibrahim et al. (11) showed that by administering a low dose of boron, the serum level of testosterone increases, and for this reason, the administration of this substance to rams improved the quality of sperm production, including their volume and movement. In the same way, Elkomy et al. (12) stated that he sought boron administration to increase testosterone, which increased the total number of sperm, their concentration, and their normal shape. Another mechanism that Özdemir et al. (20) has stated for these positive changes in the reproductive system is the antioxidant property of boron. Yildiz et al. (21) also claimed that the administration of this substance at a dose of 200 mg/kg in the drinking water of rams inhibits the production of free radicals. Other researchers believe that the beneficial effects of boron are due to strengthening the activity of serine protease inhibitor proteins (22). This protein is responsible for protecting spermatogenesis by inhibiting microbial activity. Boron also increases the immune system by increasing the activity of gamma interferon, which improves the process of spermatogenesis (23).

The reviewed studies presented diverse and contrasting results regarding the effects of boron on spermatogenesis. Some studies reported positive outcomes, indicating improvements in semen analysis parameters. For instance, Elkomy et al. (12) administered boron at different doses to rats and observed increased sperm movement, concentration, and total volume of sperm compared to the control group. Similarly, Ibrahim et al. (11) found that a diet containing boron improved total sperm volume and concentration in Osemi rams. Krishnan et al. (13) study on goats also revealed a significant increase in sperm motility after boron administration.

Several studies indicated detrimental effects of boron on the reproductive system. Ayranci et al. (15) observed increased testicular interstitial tissue edema and apoptotic cell proliferation in rats exposed to high doses of boric acid. El-Dakdoky et al. (16) study on Wistar rats showed destruction of various germ cells with increasing doses of boron. Lee et al. (10) research demonstrated that higher doses of boron led to germ cell destruction, reduced seminiferous tubule diameter, and testicular atrophy in rats.

The conflicting results observed in the reviewed studies suggest that the effect of boron on spermatogenesis is complex and may depend on factors such as dosage, duration of exposure, and animal species. These variations could explain the discrepancies between the studies. Additionally, it is important to consider the different methods and protocols used in each study, which may have contributed to the divergent outcomes. It should be noted that the studies conducted on animals may not directly translate to human results. Animal models can provide insights into potential effects, but further research is necessary to determine the impact of boron on human spermogram parameters.

The main limitations of this review were the diverse protocols and methods used in the included studies. As the outcomes of the included studies were diverse or heterogeneous, combining the data and conducting a meta-analysis was not possible. In these instances, the systematic review focuses on qualitatively summarizing the findings, identifying patterns, and discussing the implications of the diverse outcomes. Although a meta-analysis may not be feasible, a comprehensive systematic review can still provide valuable insights into the research field by highlighting gaps in knowledge, suggesting future research directions, and facilitating evidence-based decision-making. Although in the current study, most of the included studies showed a negative effect, the findings supported the conclusion that treatment with boron caused testicular toxicity, which was characterized by a dose-dependent reduction in epididymal sperm counts at higher doses and decreased spermiation at lower doses. Investigations on boron’s impact on the reproductive system have revealed that at large dosages, it can be cytotoxic. A minor link was found between blood boron levels and the average number of DNA strand breaks in spermatozoa in a zone of boric acid/borate production in Bandırma, Turkiye. Another study found that while boron compounds are not genotoxic even at the highest concentrations, they do produce oxidative stress when used in increasing amounts. Another limitation was that a great number of the studies in this subject were conducted before 2000, and it was not appropriate to exclude them. Therefore, given the potential changes in experimental standards and protocols (i.e. testicular immunohistochemistry and fluorescent immunohistochemistry staining along with most of the high-power field microscopes), these changes may have affected our conclusion.

Conclusion

The systematic review highlights the contrasting findings regarding the effect of boron on spermogram parameters. The study’s findings will help researchers better understand the limitations of boron toxicity. While some studies suggest potential benefits of boron supplementation on spermatogenesis, others indicate harmful effects. The conflicting results emphasize the need for further research to establish clear guidelines on the appropriate dosage, duration, and safety of boron supplementation in improving sperm quality and fertility. Understanding the underlying mechanisms of boron’s effects on spermatogenesis is crucial for addressing male infertility and developing targeted interventions in the future.

Authorship Contributions

Surgical and Medical Practices: A.S., An.S., S.S., M.B., M.E., A.E., F.T.A., H.S-P., S.H., M.N.B., Concept: H.S-P., M.N.B., Design: H.S-P., S.H., Data Collection or Processing: A.S., An.S., S.S., F.T.A., H.S-P., S.H., Analysis or Interpretation: An.S., S.S., F.T.A., A.E., Literature Search: A.S., M.B., M.E., Writing: A.S., An.S., M.B., M.E., M.N.B., A.E.

Conflict of Interest: No conflict of interest was declared by the author.

Financial Disclosure: The author declared that this study received no financial support.

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