Paternal contributions to telomere reprogramming in fetal development

Abstract

Objectives: Telomere length, the markers of biological aging, can be influenced by risk factors that may lead to health issues like chronic non-communicable diseases. This study explored the paternal telomere length influenced by diseases like diabetes and hypertension with age and its association with newborn telomere length (TL) and the telomerase gene. Methods: In this cross-sectional study, 204 father-newborn dyads were recruited. The qPCR was used for the quantification of TL (T/S ratio), and Sanger sequencing was done for telomerase (TERT) genotype identification. Statistical Package for Social Sciences (SPSS) and GraphPad Prism Software were used for data analysis. The Spearman correlation was used to find an association between father and newborn TL. The Kruskal-Wallis and Mann-Whitney test was used to find differences among disease groups and TL. The P<0.05 was considered statistically significant. Results: A positive correlation (r=0.39) (P<0.0001) was seen among fathers and newborn TL. The mean TL (T/S ratio) was found to be longer in healthy fathers and their newborns (1.67±1.18, 2.36±1.39), whereas significantly shorter TL (1.41±0.98, 2.02±1.58) (P=0.000) was seen in fathers suffering from diseases. The healthy fathers and their newborns TL (2.94±0.72, 3.10±0.61) were seen longer in the 15-20 (yrs) age category. Moreover, newborns of fathers (41-45 yrs) with both diabetes and hypertension had significantly longer telomere length (3.20±2.92) compared to their fathers (1.15±1.65) (P=0.006). The TERT risk genotype AC (rs2736100) was the most prevalent among the newborn girls. Conclusion: A positive association with shorter TL in newborns was found in fathers having chronic diseases such as diabetes and hypertension, highlighting the contribution of fathers in reprogramming newborns’ telomere biology.

Introduction

Advancing age and exposure to diseases that induce oxidative or cellular stress significantly reduce telomere length [1]. Shorter telomere length (TL) of leukocytes has been associated with different non-communicable diseases like Type 2 diabetes (T2DM), insulin resistance, impaired glucose tolerance, and hypertension [2-5]. The GGG sequence of telomeric DNA is prone to oxidative damage due to free radicals or reactive oxygen species, causing DNA damage, which may cause single-strand breaks [6]. The enzyme, telomerase is a reverse transcriptase that maintains TL by adding telomeric DNA repeats to chromosome ends, counteracting shortening from cell division. It comprises the TERT gene (chromosome 5p15.3, 16 exons), the major catalytic unit. Active in stem, embryonic, and cancer cells, telomerase is typically less expressed in somatic cells, except during tissue repair or actively dividing cells [7]. Moreover, the deficiency of the telomerase subunit reduces the proliferative capacity of pancreatic β-cells, contributing to impaired insulin secretion and glucose intolerance. In addition, chronic hyperglycemia increases oxidative stress, which can suppress leukocyte telomerase activity, potentially leading to telomere shortening [8,9]. Mutations or polymorphisms in TERT can dysregulate telomerase, leading to aberrant activation, disrupted cell cycles, impaired DNA repair, and oncogenesis. Dysfunctional telomerase is thus pivotal in both aging-related decline and disease progression [10].

According to the World Health Organization (WHO), the high prevalence of diabetes [11] and hypertension [12] in Pakistan highlights the importance of exploring the potential for genetic transmission. While several studies have investigated the effects of these diseases on maternal TL, as well as fetal programming and TL transmission to newborns [13,14], there is limited data on paternal transmission and its impact on newborn TL. In this context, examining scientific evidence supporting the relationship between oxidative damage, TL dynamics, and aging is crucial, as it may play a significant role in disease development in males and its potential transmission to offspring.

Considering noncommunicable diseases like diabetes and hypertension, there is no data regarding such diseases in fathers’/paternal telomeres genetics and their effect on newborns. Therefore, this study aimed to assess the influence of fathers’ diseases (diabetes and hypertension) on the telomere length of fathers and their association with newborn TL genetics.

Method

This cross-sectional study recruited n=204 father-newborn dyads from Ziauddin Hospitals. The blood samples were collected from September 2021 to July 2022 after taking patient consent. The study was approved by the university Ethics Committee “Ziauddin University Ethics Review Committee” (Ref No. 3950721SFBC). Males aged 18-45 years and their newborns, with known comorbidities such as diabetes and hypertension, were included in this study. However, individuals with a history of smoking, drug addiction, or any known malignancies were excluded. The questionnaire was filled out for demographics, occupation, education and socioeconomic status (SES), which was established by using the income of the participants (dollar rate 1 May 2022) [15]. The five ml of father’s venous blood and five ml of umbilical cord blood (venous) were collected after delivery in ethylenediaminetetraacetic acid (EDTA) tubes. The blood was then kept at 4°C, or immediately DNA extraction was done by “Qiagen DNA Blood Mini Kit (Catalog No.51306, Germany)” and stored at -20°C for further analysis.

Telomere length quantification by qPCR

The TL of blood leukocyte was quantified by “quantitative Polymerase Chain Reaction (qPCR)” as done previously [16]. The pooled blood from four healthy males and females was used as reference DNA to form a standard curve in all qPCR reactions. The DNA of the father and cord blood was used for quantification (nanograms) of telomere (T) and beta-globin gene (S) by qPCR using a master mix (Maxima Syber green: Catalog No. K0221 Thermo Scientific, USA). Each father and cord blood sample were run in triplicate. The telomere to beta-globin ratio, known as the T/S ratio, was used to calculate leukocyte TL [16,17]. The T/S ratio was determined by dividing the amount of standard DNA in nanograms (T) that corresponds to the father-newborn (experimental) sample’s copy number of the telomere template divided by the amount of standard DNA in nanograms (S) that corresponds to the copy number of the human beta-globin (single copy gene).

Sanger sequencing for TERT gene polymorphism

Sanger sequencing (n=45) of the TERT gene polymorphism was performed on father-newborn dyads, with fifteen pairs from each disease group, following the previously established method [17]. The TERT gene SNP rs2736100 (MAF >5% in Pakistanis) was amplified via PCR (544 bp product) using specific primers. The products were purified by ExoSap-IT PCR Product Cleanup kit (Catalog no. 78200, Thermo Fisher Scientific) and a Big Dye Terminator Sequencing Kit (Catalog no. 4337456, Thermo Fisher Scientific) was used for sequencing.

Data analysis

Two software programs were used for data analysis: SPSS (Statistical Package for Social Sciences, version 24) and GraphPad Prism (version 8). Quantitative variables were expressed as mean ± standard deviation (SD), while qualitative variables were summarized using frequencies and percentages. Spearman’s correlation was used to assess the relationship between the father’s and the newborn’s telomere length (TL). The Mann-Whitney U test was applied to determine differences in TL between healthy and diseased fathers. Differences in TL across various age categories and disease groups were analyzed using the Kruskal-Wallis test. Regression analysis was used to assess the association between newborn TL and paternal characteristics. A p-value <0.05 was considered statistically significant.

Results

The study included father-newborn dyads with a mean paternal age of 34±6.36 years in the healthy group (n=121) and 34±4.32 years in the disease group (n=83). The disease group included fathers with diabetes, hypertension (HTN), or both.

The mean TL (T/S ratio) was significantly longer in healthy fathers and their newborns (1.67±1.18 and 2.33±1.37, respectively) compared to diseased fathers and their newborns (1.41±0.98 and 2.02±1.58, respectively), with a statistically significant difference (P=0.000) (Figure 1).

Figure 1.

Diseased and healthy father and their newborn. The figure showed significant results in the diseased group compared to healthy group. ns: non-significant, *: significant.

In terms of socioeconomic status (SES), the highest frequency in the healthy group was observed in the upper-middle SES (41 participants, 34%), while in the disease group, the highest frequency was in the high SES category (31 participants, 37%). Regarding educational attainment, the majority of healthy fathers were graduates (36, 30%), whereas a high frequency of disease was seen in fathers with only secondary education (30, 36%). The profession with the highest disease frequency was business (32, 39%) (Table 1).

Table 1.

Demographic data of healthy fathers and those with different diseases

Variables n=204	Healthy n=121	Disease n=83
Age (mean ± SD)	34±6.36	34±4.32
Socioeconomic Status n (%)
Low	24 (20)	27 (32)
Lower middle	36 (30)	15 (18)
Upper middle	41 (34)	10 (12)
High	20 (16)	31 (37)
Education n (%)
No schooling	16 (13)	13 (16)
Secondary	26 (21)	30 (36)
Higher Secondary	15 (12)	11 (13)
Graduation	36 (30)	19 (23)
Masters	12 (10)	10 (12)
Occupation n (%)
Labourer	13 (11)	15 (18)
Private Job	73 (60)	24 (29)
Business	19 (16)	32 (39)
Other	16 (13)	12 (14)
Telomere Length (Mean ± SD)
Father	1.67±1.18	1.41±0.98
Newborn	2.33±1.37	2.02±1.58

A significant negative correlation was found between paternal age and paternal TL (r=-0.194, P=0.007) (Figure 2A). In contrast, a significant positive correlation was observed between paternal age and newborn TL (r=0.183, P=0.008) (Figure 2B). Linear regression analysis was conducted to assess the contributing factors to newborn TL. In Model 1 (P<0.25), age, paternal TL, and disease status were included. Model 2 (P<0.05) retained significant variables from Model 1: paternal age (B=0.043; P=0.009), combined diabetes and hypertension (B=-0.785; P=0.045), and paternal TL (B=0.388; P=0.000). This indicates that for every one-unit increase in paternal TL, there is a corresponding 0.38-unit increase in newborn TL (data not shown).

Figure 2.

Correlation analysis between father age and father, newborn TL. A: Analysis showed negative correlation between father telomere length (TL) and father age. B: Newborn TL and father age highlighted a positive correlation with significant results.

Figure 3 illustrates that newborn TL increased with paternal age, with the longest TL observed in newborns of fathers aged 41-45 years. However, the longest paternal TL was seen in the youngest age group (15-20 years).

Figure 3.

Father age and newborn telomere length variation. Longer newborn TL is seen in older fathers (41-45 years) compared to fathers with <40 years of age. F: father, N: Newborn.

Table 2 presents the TL differences between healthy and diseased fathers and their newborns across age categories. In the 15-20-year age group, only healthy fathers and their newborns were present, showing longer TL (2.94±0.72 in fathers and 3.10±0.61 in newborns). In the 21-25-year group, most participants were healthy, also showing longer TLs (2.49±1.22 in fathers and 2.99±1.32 in newborns). Shorter TLs were observed in fathers with diabetes and hypertension (0.74±0.78) in the 26-30-year group (P=0.76). Similarly, in the 31-35 and 36-40-year groups, shorter TLs (0.58±0.84 and 1.24±0.73, respectively) were also observed in the diabetes + HTN group, though these results were not statistically significant (P=0.54 and P=0.09, respectively). Notably, there were no healthy males in the 41-45-year age group.

Table 2.

Difference between healthy and diseased fathers and newborn TL in different age ranges

Father age (yrs)	n (%)	Telomere Length (Mean ± SD)								P value
		Healthy n=121		Diabetes n=17		Hypertension n=21		Diabetes & Hypertension n=45
		Father	Newborn	Father	Newborn	Father	Newborn	Father	Newborn
15-20	4 (2)	2.94±0.72	3.10±0.61	N/A	N/A	N/A	N/A	N/A	N/A	N/A
21-25	15 (7)	2.49±1.22	2.99±1.32	1.02±0.39	2.10±1.09	N/A	N/A	2.18±1.36	1.57±0.54	0.08
26-30	63 (31)	2.16±1.16	2.86±1.28	1.01±0.83	1.91±1.60	1.39±0.38	1.62±1.20	0.74±0.78	1.65±0.90	0.76
31-35	63 (31)	1.80±1.20	2.94±1.45	0.94±0.76	2.54±2.24	1.20±0.91	2.54±1.04	0.58±0.84	2.40±2.41	0.54
36-40	43 (21)	1.35±0.93	2.96±1.46	0.88±1.36	2.46±2.32	1.20±0.27	2.31±1.10	1.24±0.73	2.44±1.87	0.09
41-45	27 (13)	N/A	N/A	0.59±1.26	3.01±2.15	1.03±0.97	2.92±1.08	1.15±1.65	3.20±2.92	0.06
p-value		0.09		0.04*		0.001**		0.006**

Kruskal Wallis test: The mean difference between diseases and father-newborn TL. N/A: Not available.

^*P<0.05;

^**P<0.01.

Across all age categories, diabetic fathers showed shorter TLs compared to other disease groups (P=0.04). Interestingly, newborns of fathers with both diabetes and hypertension exhibited the longest TL (3.20±2.92), with statistically significant results (P=0.006) (Table 2).

Figure 4 presents the Sanger sequencing analysis of the TERT gene across the three disease groups. It shows that the heterozygous AC genotype was more prevalent in newborn girls with diseased fathers, whereas the homozygous CC genotype was more common in boys.

Figure 4.

Sanger sequencing of the TERT gene polymorphism restriction site rs2736100 (position 1286401). The black box shows the SNP C/A allele. A: The heterozygous genotype AC is more frequent in newborn girls. B: The homozygous genotype CC is more in boys to diseased fathers.

Discussion

In the current study, for the first time, the impact of paternal disease on newborn telomere length (TL) was observed. It was found that older fathers (aged 41-45 years) with both diabetes and hypertension had significantly shorter TL (0.58±0.84; P=0.006) compared to those with either condition alone. A meta-analysis similarly reported significant telomere variation in patients with hypertension [13]. However, another study showed a nonlinear association between telomere length and elevated blood pressure [5], which aligns with our current findings (P>0.05).

Age and telomere length

A significant negative correlation was observed between paternal age and TL. Typically, TL declines with advancing age, particularly in leukocytes, where shorter telomeres accelerate cellular senescence. However, this study found that older fathers were associated with longer TL in their newborns. This finding aligns with Eisenberg’s research, which suggests in utero reprogramming of telomeres [18].

Consistent with our previous studies on maternal TL, newborns exhibited longer TL than their mothers [19,20]. Similarly, a positive association between paternal and newborn TL was observed in this study. Furthermore, TL was generally longer in healthy fathers and their newborns, and shorter in both when the father was affected by disease.

Other studies have also demonstrated that increased paternal age is predictive of longer TL in offspring (P=1.84×10^-6; P=0.000) [21,22], and that sperm TL positively correlates with age. This may contribute to the transgenerational inheritance of telomere length. One explanation is that spermatogonia with shorter TL undergo apoptosis, while those with higher telomerase activity survive and produce sperm with longer TL [18,23].

Additionally, studies in humans have shown a significant relationship between offspring TL and the age of reproductive partners [18], suggesting that longer TL inheritance may result from older parents [24,25]. Telomere length has been identified as a heritable trait (β=0.14, P=1.99e-05), with strong evidence from multiple studies supporting TL transmission from parental germ cells to offspring, despite telomere reprogramming during embryogenesis [25-27].

Diseases and telomere length

TL is known to correlate with the onset and progression of age-associated diseases such as cardiovascular disease, type 2 diabetes, cancer, and neurodegenerative disorders like Alzheimer’s [28,29]. The influence of disease on telomere attrition may arise from increased oxidative stress, which disrupts glucose regulation, particularly in diabetes. While shortened TL may reduce cellular repair and replication capacity, which may lead to morbidity and mortality [30].

Previous studies have linked maternal conditions like preeclampsia and anemia with adverse pregnancy outcomes, including fetal macrosomia, malformations, miscarriage, stillbirth, and hypertensive complications, emphasizing the role of maternal health [31]. Likewise, this study suggests that paternal health can also significantly influence newborn TL.

Mechanistically, oxidative stress and reactive oxygen species (ROS) can directly damage telomeres, causing genome instability. In this study, fathers with both diabetes and hypertension showed reduced TL, likely due to oxidative damage at the molecular level. This stress induces lipid and DNA alterations, inflammation, and possibly other morbidities. As these illnesses progress, telomere shortening contributes to cell death, aging, and eventual mortality [8].

Moreover, epigenetic modifications influenced by environmental factors (e.g., social status) may alter telomere biology and trigger disease states that are transmissible to offspring [31]. Some previous studies have also linked clinical and genetic factors to the progression of non-communicable diseases like diabetes [32]. For instance, one study found a significant association between shorter TL and increased glycemic progression risk, suggesting TL may serve as a biomarker for type 2 diabetes [14].

Our study also found that diabetic fathers had shorter TL than those with only hypertension. At the molecular level, diabetes may cause β-cell dysfunction due to oxidative stress and glucose imbalance. Glucotoxicity leads to apoptosis and reduced expression of insulin-secretory components, further contributing to telomere attrition and disease progression [33].

Additionally, this study found that paternal disease-related telomere shortening in newborns may stem from vascular endothelial ROS overproduction and impaired nitric oxide-dependent vasodilation, which could predispose these children to hypertension [4].

Telomerase TERT gene

Telomerase (TERT) gene polymorphisms play a crucial role in the inheritance and regulation of TL. In this study, analysis across three paternal disease groups showed that the heterozygous AC genotype was more prevalent in newborn girls, whereas boys more frequently carried the CC genotype. The higher prevalence of the AC genotype in the disease group aligns with previous findings [9,34,35]. Variants in TERT and TP53 genes disrupt DNA repair pathways, facilitating uncontrolled cell proliferation. Notably, the AC genotype shows a stronger link to diabetes than homozygous forms [36].

Research also indicates that newborn girls are more likely than boys to inherit disease-related TL patterns from their fathers. However, no prior studies have explored gender-based differences in genotypes among newborns of diseased fathers, marking a novel finding in this study.

Limitations

Due to the limited sample size and budget constraints, only one telomerase gene restriction site was analyzed using Sanger sequencing, which may have limited the detection of broader genetic variations associated with the condition. Furthermore, the incidence of disease could not be comprehensively assessed. Additionally, there is a possibility of recall bias, as some participants may have had difficulty accurately recalling their medical history while completing the questionnaire.

Future directions

Further research with larger sample sizes, including diverse paternal disease groups, is essential to monitor telomere attrition more comprehensively. Additionally, paternal lifestyle and epigenetic factors should be explored, as telomeres serve as cellular timekeepers of biological aging. Such studies may help in mitigating the progression of non-communicable diseases like diabetes and hypertension, improving healthcare outcomes, and limiting transgenerational disease transmission.

Conclusion

A significant positive association was found between paternal and newborn telomere lengths. Notably, newborns of fathers with chronic diseases such as diabetes and hypertension exhibited shorter TL. However, newborns of fathers with both diabetes and hypertension had significantly longer telomere length compared to their fathers. This suggests that paternal disease can influence the telomeric reprogramming of offspring, highlighting the importance of paternal health in shaping transgenerational genetic outcomes.

Disclosure of conflict of interest

None.