Relationship between Semen NAD+ Concentration and Reproductive Aging in Normazoospermia Men: A Cohort Study

doi:10.21203/rs.3.rs-961019/v1

Background

The mechanisms of age-dependent reproductive decline in men are largely overlooked. Age-dependent reduction of nicotinamide adenine dinucleotide (NAD+) levels has been reported in multiple somatic and female reproductive tissues, including oocytes and ovarian tissue. However, the relationship between NAD+ levels and male zygote has not been studied yet. In the current study, the association between semen NAD+ levels and paternal age will be investigated. Besides, we will also answer if semen NAD+ levels are related to semen quality.

Methods

In this pilot observatory cohort study, semen samples of a total of 51 male subjects who visited a university-affiliated reproductive medical center for preconception consultation (<30 years: n=13, 30-40 years: n=19, >40 years: n=19) were recruited. Anthropometric characteristics were recorded, and semen analysis was performed. Sperm NAD+ levels were evaluated spectrophotometrically.

Results

Between the three age groups, there was on significant difference in major parameters of semen quality. Semen NAD+ level was also similar between the three groups (<30 years: 91.61±15.59 nmol/10⁶ sperm, 30-40 years: 125.60±16.28 nmol/10⁶ sperm, >40 years:115.59±16.55 nmol/10⁶ sperm). Additionally, linear aggression also revealed no correlation between semen NAD+ concentration and the age of the participants (r²=0.018, p=0.35). Noticeably, negative correlation was found between semen NAD+ concentrations and sperm quality parameters, including sperm concentration (r²=0.78 p<0.0001), sperm count (r²=0.47. p<0.0001.), mobile sperm number (r²=33. P<0.0001), and DFI (r²=0.35 p<0.0001). As for semen volume and mobility rate, these two parameters were not related to semen NAD+ concentration.

Conclusions

Unlike decreased NAD+ levels in aged oocytes and ovarian tissue, semen NAD+ concentration is not age-dependent. Semen NAD+ level negatively correlates with sperm quality, suggesting the unique role of NAD+ in spermatogenesis, which warrants further study and opens opportunities for pharmaceutical interventions of oligospermia.

Urology & Nephrology

nicotinamide adenine dinucleotide

sperm

age

sperm quality

DNA fragmentation index

In recent years, the trend in parenthood at an older age is becoming more noticeable, especially in modern societies. Couples intentionally delayed their marriage and children bearing to obtain additional education or to build a career. For instance, the average age of first-time fathers has risen from 27.4 years in 1972 to 30.9 years in 2017 in the U.S., with nine percent of fathers over 40 years old. Such sociodemographic change has had a profound influence on contemporary fertility. It is well acknowledged that age-dependent ovarian reserve decrease is a significant factor causing infertility in modern society, as female gamete quality declines more quickly. People gradually have turned their focus on the contribution of male aging onto infertility. Besides decreased sexual functioning and coital frequency, it is well-established that parental age profoundly impacts male fertility [1]. It is consistently reported that advanced paternal age is associated with prolonged time to pregnancy and declined the chance of pregnancy, even after adjusting the age of their wives. As for semen parameters, much evidence has suggested that semen parameters, including motility, morphology, and volume, are negatively influenced by increased parental age [2]. Notably, in couples receiving assisted reproductive technique (ART) treatment, meta-analysis also suggested a deleterious effect of increasing male age on the outcome of ART that clinical pregnancy rates and live birth rate was found to be decreased significantly when male age was >40 years [3].

While most scientific endeavor has focused on female reproductive aging, the mechanisms of age-dependent reproductive impairment in men are largely overlooked. Recent studies have revealed a new aging marker of female reproductive aging, the age-dependent decline in nicotinamide adenine dinucleotide (NAD+) in oocytes and ovarian tissue [4][5]. Importantly, these studies also opened pharmaceutical opportunities that restoration of NAD+ levels by supplementing NAD+ precursors can rescue oocyte quality, embryo development, and functional fertility in aged female mice. NAD+, the precursor of the pyridine nucleotide family, basically has two functions, a critical electrons-transfer intermediate that fuels redox reactions and a substrate for various oxidoreductase enzymes [6]. NAD+ has been a popular target for aging studies because of its pivotal role in numerous cellular processes, including DNA repair, cellular energy balancing, epigenetic homeostasis, and gene expression [7]. Studies have shown that environmental stressors can impact the reduction-oxidation status of NAD+ and consequently results in pathological changes in neurodegenerative and cardiovascular diseases [8][9][10]. This coenzyme is commonly found in biochemical pathways and all live tissues, including spermatogenesis tissues [11]. However, the role of NAD+ in spermatogenesis and age-dependent reproductive impairment has not been well established yet. Considering the importance and potential therapeutic value of NAD+ in female reproductive aging, the relationship between levels of sperm NAD+ and male aging will be investigated in this pilot study.

Study population and data acquisition

This study was approved by the Ethical Committee of Beijing Chao-Yang Hospital (Ethical Approval No. 2021-ke-458). All the participants of the current study are enrolled from the reproductive medical center of Beijing Chao-Yang hospital from July to September 2021. They were husbands of secondary infertile couples who had a previous birth but failed to conceive after one year of unprotected intercourse. Informed consent was obtained from each participant. Their semen samples were collected for semen analysis as a routine fertility examination. A total of 61 participants aged 22-60 years old were all in good mental and physical state. The conditions which may cause dysspermia were excluded, including chronic diseases, urogenital infections, varicocele, oligospermia, and azoospermia. Their general information, including body mass index (BMI), education levels, smoking status, and abstinence period, was collected through a questionnaire.

Semen collection and analysis

The semen specimens were collected by masturbation after 2–7 days of sexual abstinence. Semen samples were masturbated into a sterile plastic container and then transferred to our andrology laboratory. Incubation at 37 °C for 30 min was performed to allow liquefaction. After liquefaction, semen volume was evaluated by weighing. Computer Aided Sperm Analysis (CASA) system was used to measure semen parameters (sperm count and motility) according to WHO guidelines [12]. For each measurement, at least 200 spermatozoa were analyzed.

Sperm chromatin integrity was assessed by the flow cytometric-based sperm degree of DNA fragmentation chromatin structure assay (SCSA, CellPro Biotech, Ningbo, China). The assays were performed according to the manufacturer’s instructions. Finally, the DNA fragmentation index (DFI) was used to report sperm chromatin integrity.

Measurement of Intracellular NAD+ levels

Intracellular NAD+ levels were measured by cycling assay as previously described [5]. Using a NAD/NADH Cell-Based Assay Kit (Cayman Chemical, MI, USA)，the cycling assay for sperm NAD+ levels was measured according to the manufacturer’s instruction. Briefly, 300 μl semen was centrifuged and transferred 96-well v-bottom plate. Each reaction was run in duplicate. After wash and lysis, 100 μl of the supernatant was transferred to corresponding wells of a new plate. After incubation with reaction solution, the absorbance of each sample was measured. The NAD+ levels were calculated in the absence and presence of NADH inhibitor using a plate reader at wavelength of 450 nm.

Statistical analysis

Categorical parameters were summarized with counts and percentages. Continuous variables were described with means and standard deviations. First, one sample Kolmogorov–Smirnov test was used to determine whether analyzed variables follow a normal distribution. If the parameter was consistent with skewed distribution (sperm number, DFI, and NAD+ concentration), these variables were log-transformed before further statistical analyses. Secondly, the chi-square test or Fisher’s test was used to analyze nominal variables, while student’s t-test was employed to analyze quantitative variables. Linear regression was used to explore the correlation between NAD+ levels and sperm quality parameters. All data analyses were conducted using SPSS 11.0 software (SPSS Inc., Chicago, IL, USA). P-value < 0.05 was considered statistically significant.

A total of 61 participants were recruited from October 2020 to January 2021. Ten out of the 61 participants were excluded for conditions including azoospermia, oligospermia, and urogenital infections. Characteristics of the 51 subjects by age category are summarized in Table 1. The mean BMI of the group older than 40 years old (24.30±0.70) was slightly lower than the other two groups (< 30 years: 25.62±0.85, 30-40 years: 26.03±0.73), while the difference did not reach statistical significance. About 72% of participants received undergraduate or higher education, and the distribution of education status was similar between the three age groups. As for smoking status, about 35% of subjects were smokers, and the percentage of smokers was similar between the age categories. The mean abstinence period was not significantly different between the three age categories (< 30 years: 4.54±0.37 days, 30-40 years: 4.21±0.29 days, >40 years: 4.53±0.29 days).

Table 1

Characteristics of study participants by age category
Characteristics	All participants		Age category
Characteristics	All participants	<30 (n=13)	30-40 (n=19)	>40 (n=19)
BMI (kg/m²)
Underweight (<18.5)	0	0	0	0
Normal (18.5-24.9)	26	6	8	12
Overweight (25-29.9)	20	5	9	6
Obese (≥30)	5	2	2	1
Education
High school and lower	14	4	4	6
Undergraduate and higher	37	9	15	13
Smoking status
Non-smoker	33	8	12	13
Smoker	18	5	7	6
Abstinence period (days)
2-3	19	4	8	7
4-5	22	6	8	8
6-7	10	3	3	4

Semen parameters and sperm kinematics between the three age categories were compared in figure 1. The analyses indicated there were no significant differences for semen volume (< 30 years: 2.85±0.42 ml, 30-40 years: 2.93±0.31 ml, >40 years: 2.97±0.33 ml), concentration (< 30 years: 72.50±7.19 10⁶/ml, 30-40 years: 62.64±6.49 10⁶/ml, >40 years: 60.84±6.63 10⁶/ml), total sperm number (< 30 years: 233.83±44.15 10⁶, 30-40 years: 187.34±33.27 10⁶, >40 years: 182.81±31.40 10⁶). Analysis of sperm kinematics also indicated no significant differences for motility rate (< 30 years: 34.80±4.00 %, 30-40 years: 34.95±2.91 %, >40 years: 27.66±2.62 %), mobile sperm number (< 30 years: 70.74±15.39 10⁶, 30-40 years: 70.29±14.97 10⁶, >40 years: 47.73±9.40 10⁶) between the age categories. Sperm samples from subjects over 40 years old had slightly lower mobility rate and motile sperm number, though the difference did not reach statistical significance. Additionally, levels of DFI were also comparable between the age categories (< 30 years: 18.75±3.30 %, 30-40 years: 22.00±2.14 10⁶, >40 years: 21.97±3.00 10⁶).

The relationship between age and NAD+ concentration was analyzed in Figure 2. The younger group (<30 years) has the lowest NAD+ concentration comparing the other two elder groups, though the difference did not reach statistical significance (< 30 years: 92.61±15.59, 30-40 years: 125.60±16.28, >40 years: 115.59±16.55). Further linear regression analysis revealed no correlation between NAD+ concentration and the age of the participants.

Next, linear regression was performed to answer if NAD+ concentration was correlated with semen parameters and sperm kinematics. Our results revealed that intracellular NAD+ level was negatively correlated with sperm quality parameters, as shown in Figure 3. Additionally, subjects with higher sperm NAD+ concentrations also had relatively elevated DFI.

This is the first observatory cohort study focusing on the relationship between spermatozoa NAD+ levels and reproductive aging in men. Our result revealed no decline in sperm NAD+ levels along with parental age. Noticeably, the intracellular concentration of NAD+ has a significant negative relationship with sperm quality parameters. In contrast with the well-documented age-dependent decline in NAD+ level during female reproductive aging, our results highlight that NAD+ has a unique role in spermatogenesis and male reproductive aging.

Age-dependent decline in semen parameters and sperm kinematics has been considered the underlying mechanism of male reproductive aging. Early studies have consistently reported a reduction in sperm motility along with advancing age. 0.17–0.6% decrease in motility per year of age was observed [13][14][15]. Semen volume decreased at a very modest rate of 0.15–0.2% per year of age. In contrast with motility and volume, the relationship between sperm concentration and age is the least consistent. Except for a reported decrease of 3.3% per year in sperm concentration [13], other population-based large cohorts revealed a 0.7% increase per year of age and an increase of 14% over a two-decade period [16]. Consistent with previous findings, the current study also suggests a tendency to decline in sperm quality along with age. Albeit limited sample size, these elder subjects generally had fewer sperms and poorer motility, as revealed by our data. As for DFI, there has been a good amount of literature showing an increasing rate of fragmentation with increasing age because of increased oxidative stress over time [17][18][19]. Our data also support that advanced age was related to a marginal elevation of DFI.

During the last decade, a growing amount of knowledge has revealed the significance of redox active nucleotides in the process of aging. Evidence has shown that in different somatic tissues level of NAD+ declines along with age. Recent studies clearly indicated that aging accompanies declined levels of NAD+ in rodent oocytes and ovarian tissue, which underlies age-related loss of oocyte function [4]. Importantly, repleting NAD+ by supplementing metabolic precursors reversed ovarian aging by improving mitochondrial functions in aged rodents, which gained attention as a potential therapeutic target for degenerative diseases [5][20].

To investigate if the age-dependent NAD+ decline can be recapitulated in males, the current study focused on male reproductive aging and measured NAD+ levels of human semen samples. Interestingly, despite our limited sample size, the results showed no apparent association between age and sperm NAD+ levels. This inconsistency of age-related alteration of NAD+ levels between oocytes and sperms can be explained by different self-renewal mechanisms of female and male zygotes. According to conventional theory, unlike most somatic tissues, oocytes of mammalian species cannot be regenerated by resident precursor stem cells. The oocyte pool is fixed during in urero development, and the oocyte population does not undergo self-renewal, making them highly vulnerable to age-related abnormalities held [21]. In stark contrast with oocytes, the male zygote is continuously regenerated thought out adult life in the seminiferous tubules. The entire process of spermatogenesis takes approximately 120 days. Thus newly generated young germ cells are not likely susceptible to age-related NAD+ decline.

This study reported a negative correlation between intracellular sperm NAD+ level and sperm quality parameters for the first time. Subjects with elevated intracellular sperm NAD+ concentration tend to have poorer semen concentration, motility, and DFI. In a recent rodent study, oral supplementation of NAD+ precursors, nicotinamide mononucleotide (NMN), increases NAD+ concentration in testes and significantly impairs sperm count and motility [22]. This phenomenon can be explained by mitochondrial function. High NAD+ levels stimulate mitochondrial biogenesis and function. ROS is a primary by-product of the NADH-to-NAD+ mitochondria conversion through a series of energy metabolism-related processes. As a result, elevated ROS and oxidative stress could result from hyperactivity of the mitochondrial. As in sperm, ROS is primarily from the mitochondrial NADH-dependent oxidoreductase reaction [23]. The high levels of intracellular NAD+ potentially exert delirious effects on sperm quality and increase DFI via elevating mitochondrial ROS outputs. Additionally, previous findings indicated that supplementation of another NAD+ precursor, nicotinamide riboside (NR), downregulates sirtuins which play a key role in spermatogenesis[24][25][26].

This is the first report focusing on the relationship between semen NAD+ concentration and reproductive aging in normozoospermic men. The next goal will be to answer if high levels of sperm NAD+ on sperm have an adverse influence on the clinical outcomes of ART. The role of NAD+ in spermatogenesis also warrants further study, which will induce us to novel therapeutic approaches to dysspermia.

ART: assisted reproductive technique

BMI: body mass index

CASA: Computer Aided Sperm Analysis

DFI: DNA fragmentation index

NAD+: nicotinamide adenine dinucleotide

NMN: nicotinamide mononucleotide

NR: nicotinamide riboside

Ethics approval and consent to participate

The Ethical Committee of Beijing Chao-Yang Hospital approved the study (Ethical Approval No. 2021-ke-458). Informed consent was obtained from each participant. The study was carried out in accordance with the Helsinki Declaration.

Consent for publication

Not applicable

Availability of data and materials

The datasets used and during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests

Funding

This work was supported by grants from the National Natural Science Foundation of China (No. 81871134).

Authors' contributions

X.B performed the experiment and drafted the manuscript. P.W conceived the idea and polished the manuscript. All authors read and approved the final manuscript.

Acknowledgments

The authors would like to thank all the patients who participated in the study.

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No competing interests reported.

Relationship between Semen NAD+ Concentration and Reproductive Aging in Normazoospermia Men: A Cohort Study

Status:

Version 1

Abstract

Figures

Background

Methods

Study population and data acquisition

Semen collection and analysis

Measurement of Intracellular NAD+ levels

Statistical analysis

Results

Discussion

Conclusions

Abbreviations

Declarations