Report on Age-Related Decline in Behavior and Reproductive Health in Male Mice: Implications for Sustainable Development Goals

Abstract

Research has traditionally emphasized the impact of advanced reproductive age on women, primarily due to biological constraints. However, the effects of paternal aging on fertility and offspring development remain underexplored. This study investigates the influence of aging on sexual behavior, sperm quality, and embryo and fetal development in male mice aged 4 months (control), 19 months, and 24 months. Findings reveal that older males exhibit reduced reproductive interest and locomotor activity, decreased serum testosterone levels, and diminished testicular weight. Sperm from aging males show increased morphological anomalies, protamine deficiency, and impaired capacitation. Embryo development rates, cleavage, blastocyst formation, and initial cell differentiation are adversely affected by paternal age. Furthermore, fetuses from aged males are smaller and lighter, with a reduced fetal weight ratio. These results underscore the multifactorial decline in male reproductive capacity with age, highlighting critical considerations for reproductive health and offspring well-being.

Introduction

Demographic shifts have led to increased paternal age at childbirth globally, with significant rises in births from fathers aged 35 to 54 years and declines in younger paternal age groups. This trend is influenced by social factors such as career progression, economic stability, and access to assisted reproductive technologies. While female reproductive aging has been extensively studied, male reproductive aging and its consequences on fertility and offspring health require further elucidation.

Age-related declines in male sexual behavior, testosterone levels, and sperm quality have been documented, with implications for reproductive success and offspring development. This study utilizes a murine model to investigate the onset and progression of reproductive senescence in males, focusing on behavioral, hormonal, sperm functional, and embryonic developmental parameters.

Methods

Experimental Design

1. Two blinded experiments were conducted using male C57BL/6J mice aged 4 (control), 19, and 24 months.
2. Experiment 1 assessed behavioral patterns, sperm parameters, and in vitro embryo development using the same animals.
3. Experiment 2 evaluated in vivo fetal development at 16 days of gestation with a separate cohort of males.
4. Females aged 2 to 3 months were hormonally synchronized for mating.

Behavioral Assessments

• Open field test to evaluate locomotor activity and anxiety-like behavior.
• T-maze alternation test for spatial memory assessment.
• Spontaneous social interaction test with synchronized females to assess reproductive interest and sexual behavior.

Reproductive and Embryo Development Assessments

• Serum testosterone levels measured via validated ELISA.
• Sperm morphology, motility, capacitation, and chromatin status evaluated using microscopy, CASA, flow cytometry, and chromomycin A3 staining.
• In vitro embryo development monitored up to blastocyst stage with immunofluorescence analysis of cell differentiation markers (SOX2 and CDX2).
• In vivo fetal development assessed by measuring fetal and placental size and weight at gestational day 16.

Results

Experiment 1: Behavioral and Reproductive Assessments

1. Behavioral Findings:
   • Aging males (19 and 24 months) showed significantly reduced locomotor activity, including decreased total distance traveled, lower frequency of zone entries, reduced rearing, and slower average speed compared to 4-month controls.
   • Sexual behavior was impaired in aged males, with reduced social interaction time and frequency of female chasing, indicating diminished reproductive interest.
   • Spatial memory remained intact across age groups.
2. Testicular and Hormonal Changes:
   • Serum testosterone levels and testicular weights were significantly lower in 19- and 24-month-old males compared to controls.
3. Sperm Quality and Function:
   • Increased sperm morphological defects, including tail and midpiece abnormalities, were observed in aging groups.
   • Protamine deficiency was higher in 24-month-old males, indicating compromised chromatin condensation.
   • Capacitation efficiency was reduced in aged males, with a lower percentage of capacitated sperm and increased premature acrosome reaction.
4. Embryo Development:
   • Cleavage, blastocyst formation, and overall embryo development rates were significantly reduced in 24-month-old males.
   • Embryos from aged males exhibited delayed kinetics, with a higher proportion of early blastocysts and lower rates of expanded and hatched blastocysts.
   • First embryonic cell differentiation was compromised, with reduced inner cell mass (ICM) and trophectoderm (TE) cell counts and decreased expression of SOX2 and CDX2 proteins.

Experiment 2: In Vivo Fetal Development

• Fetuses sired by 24-month-old males were significantly smaller and lighter, with a reduced fetal-to-placental weight ratio compared to controls.
• No significant differences were observed between 19-month-old males and controls in fetal or placental parameters.
• Litter size characteristics remained unchanged across groups.

Discussion

This study provides comprehensive evidence that paternal aging adversely affects male reproductive behavior, hormonal status, sperm quality, and subsequent embryo and fetal development. The observed decline in sexual behavior and testosterone levels aligns with known age-related hormonal changes, impacting reproductive success. Sperm morphological defects and impaired capacitation contribute to reduced fertilization efficiency and compromised embryo quality.

Embryonic developmental delays and impaired cell differentiation observed in embryos from aged males may underlie the reduced fetal growth and weight, with potential long-term consequences for offspring health. These findings emphasize the importance of considering paternal age in reproductive health assessments and interventions.

Implications for Sustainable Development Goals (SDGs)

This research aligns with several United Nations Sustainable Development Goals, particularly:

• SDG 3: Good Health and Well-being – By elucidating the impact of paternal aging on reproductive health and offspring development, this study supports efforts to improve maternal and child health outcomes and reduce adverse pregnancy outcomes.
• SDG 5: Gender Equality – Expanding the focus beyond maternal factors to include paternal reproductive health promotes a more comprehensive understanding of reproductive rights and health.
• SDG 10: Reduced Inequalities – Understanding biological and social factors influencing reproductive aging can inform equitable access to reproductive technologies and healthcare services for older parents.
• SDG 12: Responsible Consumption and Production – The use of animal models in research adheres to ethical guidelines promoting responsible scientific practices.
• SDG 17: Partnerships for the Goals – The collaborative nature of this research, involving multiple institutions and funding bodies, exemplifies partnerships essential for advancing scientific knowledge and health outcomes.

Conclusion

Advanced paternal age in male mice leads to a multifactorial decline in reproductive behavior, sperm function, and embryo and fetal development. These findings highlight the need for increased awareness and research on paternal factors in reproductive health, contributing to the achievement of Sustainable Development Goals related to health, equality, and responsible research practices.

1. Sustainable Development Goals (SDGs) Addressed or Connected

1. SDG 3: Good Health and Well-being
   • The article focuses on reproductive health, aging, and fertility, which are critical components of ensuring healthy lives and promoting well-being at all ages.
   • It addresses issues related to male reproductive aging, hormonal decline, sperm quality, embryo development, and fetal outcomes.
2. SDG 5: Gender Equality
   • The article highlights the importance of understanding male reproductive aging, which has traditionally been less studied compared to female reproductive aging, thus contributing to gender-inclusive health research.
3. SDG 10: Reduced Inequalities
   • The research addresses demographic changes and social factors influencing delayed fatherhood, which relate to inequalities in reproductive health access and outcomes.
4. SDG 9: Industry, Innovation and Infrastructure
   • The article mentions the use of assisted reproductive technologies, which are part of innovative health technologies improving reproductive outcomes.

2. Specific Targets Under the Identified SDGs

1. SDG 3: Good Health and Well-being
   • Target 3.7: Ensure universal access to sexual and reproductive health-care services, including for family planning, information and education, and the integration of reproductive health into national strategies and programs.
   • Target 3.4: Reduce by one third premature mortality from non-communicable diseases through prevention and treatment and promote mental health and well-being (relevant to aging and reproductive health).
2. SDG 5: Gender Equality
   • Target 5.6: Ensure universal access to sexual and reproductive health and reproductive rights as agreed in accordance with the Programme of Action of the International Conference on Population and Development and the Beijing Platform for Action.
3. SDG 10: Reduced Inequalities
   • Target 10.2: Empower and promote the social, economic and political inclusion of all, irrespective of age, sex, disability, race, ethnicity, origin, religion or economic or other status.
4. SDG 9: Industry, Innovation and Infrastructure
   • Target 9.5: Enhance scientific research, upgrade the technological capabilities of industrial sectors, including health technologies.

3. Indicators Mentioned or Implied to Measure Progress

1. Indicators related to SDG 3 (Good Health and Well-being):
   • Serum testosterone levels as a biomarker for male reproductive health and aging.
   • Sperm quality parameters including morphology defects, protamine deficiency, capacitation efficiency, and DNA fragmentation.
   • Embryo development rates: cleavage rate, blastocyst formation rate, embryo development rate, and blastocyst stage progression.
   • Fetal development indicators: fetal size, fetal weight, and fetal-to-placental weight ratio.
   • Behavioral assessments related to sexual behavior and locomotor activity as indirect indicators of reproductive health.
2. Indicators related to SDG 5 (Gender Equality):
   • Inclusion of male reproductive aging studies to balance gender-focused reproductive health research.
3. Indicators related to SDG 10 (Reduced Inequalities):
   • Demographic data on paternal age trends and birth outcomes related to advanced paternal age.
4. Indicators related to SDG 9 (Industry, Innovation and Infrastructure):
   • Use and success rates of assisted reproductive technologies (IVF/ICSI) as indicators of technological advancement in reproductive health.

4. Table of SDGs, Targets, and Indicators

Source: nature.com