Evaluation of telomere length and telomerase activity on predicting in vitro fertilization treatment outcomes

IVF includes 4 crucial steps: ovarian stimulation, oocyte retrieval, embryo fertilization, and embryo transfer [ 7 ]. During these procedures, gametes and embryos are exposed to numerous factors and chemicals, which may induce mitochondrial, genetic, and epigenetic alterations on TL and TA in IVF-derived gametes and embryos, with potential impact on their quality [ 59 ]. Regarding ovarian stimulation, several studies have reported that superovulation can cause alterations in the epigenome of oocyte and embryos; however, studies are needed to evaluate how ovarian stimulation protocols influence TL and TA of oocytes and embryos [ 59 ]. Oocytes retrieved are then fertilized with the collected sperm. A growing evidence demonstrates that manipulations of sperm samples during IVF (handling, separation method, washing techniques, cryopreservation) could alter paternal epigenome or sperm trancriptome and could impair fertilization, embryo quality, and IVF outcome [ 60 , 61 ]. Several reports have suggested that in vitro culture period and conditions, such as culture media, oxygen levels, temperature, humidity, osmolality, and pH, which mimic human organism during IVF process, can influence implantation, as well as pregnancy rates due to their effect on embryo quality [ 5 , 59 – 62 ]. Recently, a limited number of studies have shown that the exposure of oocytes and embryos to culture conditions may increase ROS amounts and influence intracellular events (e.g., epigenetic modifications), which may trigger changes of telomere length in the early embryos [ 5 , 59 – 62 ]. Another interesting factor is the increased BMI in females and in combined females and males, which is associated with lower numbers of available embryos, high-quality embryos, and decreased fertilization rates in the female group with increased BMI [ 63 ]. However, only one study has shown that high maternal BMI is associated with shorter LTL and activation of telomere shortening and this was linked with a poor IVF outcome [ 5 , 64 ]. Moreover, exogenous and mainly environmental factors (pollution, exposure to toxic agents, pesticides, or herbicides) may indirectly affect embryo TL and IVF outcome [ 5 ]. Additional studies are required to elucidate potential negative impact of these factors on telomeres.

It is now well established that the fertilizing spermatozoon contributes numerous factors which interact with their female counterparts and has a dynamic and critical participation in normal fertilization and postfertilization developmental steps [ 35 ]. A deeper comprehension of the contributions of paternal components and the assurance of sperm quality are crucial for ART success [ 35 ]. Thus, it is of great importance the identification of a novel diagnostic and prognostic biomarker for the evaluation of sperm quality and function [ 36 ]. Accumulating data have demonstrated that sperm TL (STL) is a new molecular marker of sperm quality [ 36 ]. Studies focused on the relation between telomere length and telomerase activity and spermatogenic cells are presented in Table  2 . Table 2 Studies focused on the relation between telomere length/telomerase activity and spermatogenic cells Studies Samples Methodology used for TL/TA measurement Main findings Kimura (2008) [ 50 ] Leukocytes from 634 men (18–94 years old) and sperm from 46 young (< 30 years) and older (50 years) donors Q-FISH - LTL in offspring was positively correlated with paternal age at the time of birth - A subset of sperm in older men had with elongated telomeres Antunes (2015) [ 49 ] 20 individual spermatozoa from 10 men undergoing IVF q-PCR TL among individual spermatozoa within an ejaculate varies markedly and increases with age Mishra (2016) [ 37 ] 112 infertile men and 102 age-matched fertile controls q-PCR Mild oxidative stress results in longer telomeres, while severe oxidative stress results in shorter telomeres Gentiluomo (2021) [ 43 ] 599 men undergoing semen evaluation q-PCR - No associations between sperm parameters and STL nor LTL were observed - Four SNPs were weakly associated with sperm variables Cariati (2016) [ 44 ] 73 samples from men (31–52 years old) q-PCR STL was found to be strongly related to sperm count, implying the theory that aneuploidy and sperm DNA fragmentation are linked Biron-Shental (2018) [ 45 ] 100 sperm cells from 16 sub-fertile and 10 fertile men Q-FISH Sub-fertile sperm cells have short telomeres that are elongated by the alternative pathway of telomere capture Berneau (2020) [ 51 ] 66 normozoospermic male partners of couples undergoing ART q-PCR STL is related to IVF rates but not to sperm characteristics or lifestyle factors Sharqawi (2022) [ 52 ] 34 men (18–60 years old) from couples undergoing IVF q-PCR A healthy lifestyle is connected with extended STL and good sperm quality in men undergoing IVF Yang (2016) [ 56 ] 306 overweight males (> 28 kg/m 2 ) and 345 age-matched normal weight individuals (20–25 kg/m 2 ) q-PCR - Couples with male BMIs > 28 kg/m 2 had poorer rates of fertilization, superior embryo development, and clinical pregnancy rate than couples with normal BMI males - Mean STL value for the overweight BMI group was significantly shorter than that of the normal BMI group Yang (2015) [ 57 ] 418 men from couples planning IVF qPCR STL was positively correlated with embryo quality and transplantable embryo rates, but not with clinical pregnancy rates TL : telomere length, TA : telomerase activity, Q-FISH : quantitative fluorescence in situ hybridization, LTL : leukocyte telomere length, IVF : in vitro fertilization, STL : sperm telomere length, q-PCR : quantitative polymerase chain reaction, SNP : single-nucleotide polymorphism, ART : assisted reproductive technology, BMI : Body Mass Index Studies focused on the relation between telomere length/telomerase activity and spermatogenic cells - LTL in offspring was positively correlated with paternal age at the time of birth - A subset of sperm in older men had with elongated telomeres - No associations between sperm parameters and STL nor LTL were observed - Four SNPs were weakly associated with sperm variables - Couples with male BMIs > 28 kg/m 2 had poorer rates of fertilization, superior embryo development, and clinical pregnancy rate than couples with normal BMI males - Mean STL value for the overweight BMI group was significantly shorter than that of the normal BMI group TL : telomere length, TA : telomerase activity, Q-FISH : quantitative fluorescence in situ hybridization, LTL : leukocyte telomere length, IVF : in vitro fertilization, STL : sperm telomere length, q-PCR : quantitative polymerase chain reaction, SNP : single-nucleotide polymorphism, ART : assisted reproductive technology, BMI : Body Mass Index Numerous studies have discovered a favorable relationship between STL and traditional indicators of high sperm quality, such as motility, vitality, and sperm count. This has also been linked to other DNA integrity-related traits, with research showing a correlation between STL and DNA fragmentation and protamination levels. Additionally, it is important to mention that telomere attrition, which is mostly linked to an oxidative stress situation, influences sperm quality and has a negative impact on reproductive potential and increased DNA instability [ 37 ]. Experiments have demonstrated that there is a link between sperm DNA damage and semen characteristics, male infertility, conception, and treatment response [ 38 , 39 ]. Telomere length also seems to be a reliable biomarker for female and male infertility since it has a significant role in fertilization outcomes at in vitro setting [ 40 ]. Vasilopoulos et al. provided an outline of general trends regarding the association of TL with infertility factors, by proving epidemiological and original research studies [ 41 ]. A favorable association between male infertility factors and shorter STL was found in the majority of the studies [ 41 ]. Amir et al. revealed that the death of spermatozoa, decreased motility, low sperm count, incorrect chromosomal pairing and movement during meiosis, and unsuccessful fertilization are some of the mechanisms through which sperm telomere shortening is linked to male infertility [ 42 ]. However, Gentiluomo et al. did not find a direct correlation between telomere and male infertility in their analyses on male spermatogenesis and infertility [ 43 ]. They found four single-nucleotide polymorphisms (SNPs) to be weakly correlated to sperm variables, suggesting that these SNPs are pleiotropic and may be involved in other regulatory mechanisms unrelated to telomere homeostasis but nonetheless involved in the spermatogenic process [ 43 ]. The findings of their research indicate that while TL is not directly associated with male infertility, the selected SNPs may still be involved indirectly [ 43 ]. In order to determine if STL variation is related to chromosomal abnormalities, DNA fragmentation, conventional semen characteristics, IVF success, or all four factors, Cariati et al. analyzed STL in semen samples from men [ 44 ]. They showed that shorter telomeres are correlated with an elevated percentage of diploidy [ 44 ]. Oligospermic samples exhibited particularly short telomeres, and STL was also discovered to be positively linked with sperm count [ 44 ]. Atypical STLs were present in 17.6% of the samples in total [ 44 ]. All these samples failed to result in a continuing pregnancy [ 44 ]. They concluded that STL has the potential to be a quick and affordable method of evaluating sperm quality [ 44 ]. Moreover, according to additional studies, males who have idiopathic infertility have significantly shorter sperm telomeres and a lack of telomere homeostasis than healthy controls, which is demonstrated by lower amounts of telomerase [ 44 ]. Similar findings were made by Biron-Shental et al. who discovered that subfertile men had less sperm cells with telomerase reverse transcriptase positivity and more sperm cells with shorter TL than controls [ 45 ]. Early spermatogenesis has been associated with elevated telomerase expression, whereas spermiogenesis, the late phase of spermatogenesis, is associated with falling telomerase expression (Fig.  1 ). This suggests that whereas spermatids and mature spermatozoa exhibit decreased telomere lengths, spermatocytes preserve their telomere length [ 45 ]. Another possibility is that numerous complex chromosome reorganization processes take place during the late stages of spermatogenesis [ 5 ]. Thus, an altered telomere dynamic in sperm may be a possible cause for a decline in male reproductive potential, especially in idiopathic instances, and points to the detrimental effects of telomere shortening on sperm function [ 46 ]. Spermatogenesis is a dynamic and continuous process in men with no critical age at which sperm production cease [ 42 ]. However, advanced paternal age (APA) has been shown to negatively affect sperm quality and testicular functions [ 47 ]. In addition, APA has been associated with epigenetic changes, DNA mutations, chromosomal abnormalities, increasing rate of preterm death, and decreased IVF success rate [ 47 ]. It is well known that STL increases with advancing age maybe due to the high telomerase activity in the testes and/or the ALT pathway [ 48 ]. Male germ cells have average TL 10–20 kb, with elongation ranging from 17 to 135 bp/year depending on the methodology used [ 36 , 42 ]. TL among individual sperm subpopulations within an ejaculate also show increasing heterogeneity with advancing age [ 49 ]. Moreover, APA at conception has been positively correlated with longer LTL and STL in offspring of older fathers, thus confirming the telomere length heritability [ 36 , 48 , 50 ]. Selection of spermatozoa with long telomeres is essential for the development of excellent quality embryos, and may even be necessary for a successful IVF outcome, according to several studies that link STL with good quality embryos [ 5 , 51 – 53 ]. Specifically, spermatozoa with longer telomeres are more likely to result in better quality embryos and possibly to a successful IVF outcome [ 5 ]. ΙVF failure and a higher likelihood of recurrent miscarriage have been linked to embryo aneuploidy, with short telomeres thought to be the primary cause of aneuploidy and delayed embryo development [ 5 ]. These evidences are endorsed by further research that showed that in normozoospermic samples, there was a positive link between sperm telomere length and fertilization rate, as well as a clear trend towards higher sperm telomere length in successful embryo implantation rates [ 51 ]. However, no link between sperm telomere length and sperm parameters was discovered and no correlation was found between sperm telomere length and lifestyle factors [ 51 ]. Overall, the findings imply that sperm telomere length may have an essential mechanistic role in fertilization rate regardless of sperm characteristics or lifestyle factors [ 51 ]. On the contrary, Sharqawi et al. examined how lifestyle choices affected sperm’s telomere length and tracked how that relationship related to IVF success [ 52 ]. They found that a good lifestyle is associated with long STL and good sperm quality in patients undergoing IVF [ 52 ]. In addition, Yuan et al. performed a meta-analysis, including 12 prospective observational cohort studies in order to assess the accuracy and clinical value of STL as a new marker for diagnosing male infertility and predicting the quality of embryonic development [ 53 ]. They revealed that embryonic aneuploidy may be linked to an elevated risk of IVF failure and recurrent miscarriage [ 53 ]. Given the fact that short telomeres are correlated with increased aneuploidy and delayed embryo development, it was hypothesized that sperm telomere length might be a promising predictor of embryonic development, both for natural conception and IVF, because it could represent embryonic quality and predict pregnancy outcome to some extent [ 53 ]. Interestingly, Van Opstal et al. displayed that in couples undergoing IVF therapy advanced male age adversely influences the likelihood of reaching the 8-cell stage at D3 [ 54 ]. They observed a significant inverse association between APA and a key determinant for scoring of embryo quality: older men were less likely to produce an embryo of eight blastomeres at D3, compared to younger fathers [ 54 ]. On the other hand, Lu et al. did not discover a relationship between male age and high-quality or transferrable embryos, instead they revealed that blastocyst formation rates were unaffected by male paternal age and normal semen characteristics [ 55 ]. In the same research, it was indicated that there were no differences in the birth weights of newborns across the various paternal age groups, and that the miscarriage rate for fathers between the ages of 35 and 39 was substantially higher than that for fathers under 35 [ 55 ]. The live birth rate was on the decline, although there was no appreciable variation between groups [ 55 ]. Yang et al. studied the effect of paternal overweight or obesity on IVF treatment outcomes and the possible mechanisms that are involved. They supported that couples with males who had Body Mass Index (BMI) exceeding 28 kg/m 2 had lower rates of fertilization, high-quality embryo development, and clinical pregnancy than couples with men who had BMIs between 20 and 25 kg/m 2 [ 56 ]. Additionally, the mean STL for each patient was calculated, and the findings revealed that the overweight BMI group’s mean value was much shorter than that of the normal BMI group [ 56 ]. Additionally, it was discovered that whereas sperm mitochondrial activity was lower in the overweight BMI group compared to the normal BMI group, sperm DNA fragmentation rate and reactive oxygen species (ROS) content in semen were higher [ 56 ]. Finally, Yang et al. found a correlation between sperm TL and the viability of IVF-created embryos, indicating that the telomeric state of male gametes may have an impact on later embryonic development [ 57 ]. To ascertain the relationships between STL, fertilization laboratory parameters, and clinical pregnancy in IVF, researchers evaluated 418 couples [ 57 ]. The mean STL for each patient was found using the quantitative PCR method after semen samples were collected [ 57 ]. They discovered that STL is positively correlated with embryo quality during IVF and suggested that STL may be utilized as a marker for the prediction of embryonic quality. However, more research is required to support these findings [ 57 ]. To conclude, even though the literature shows conflicting evidence on the effect of STL on semen quality, the majority of studies imply that STL can be an effective biomarker for male infertility and possibly help in the improvement of the success rate of fertility treatments [ 58 ]. The conflicting findings on STL in semen as a fertility marker could be due to limitations of the study, such as small size, different populations studied, and different techniques used. Thus, it is of great value for further studies to take place and define whether STL is an appropriate biomarker for male infertility and whether it could be a valuable tool for assisted reproduction [ 58 ].

Mounting evidence suggests a close relationship between telomeres and telomerase and reproductive aging. Although a growing number of studies have proposed a positive association between telomeres and IVF, the data is limited and further research is needed to shed more light into their role in IVF. Given that telomere length and telomerase analysis is a reasonably simple and inexpensive approach, it may meet the criteria for its inclusion in the IVF procedure as a prognostic biomarker for determining embryo quality, as well as pregnancy success.

Telomeres are specialized nucleoprotein structures found at the end of linear chromosomes. In humans, telomeres are composed of tandem TTAGGG repeats bound by a specialized protein complex known as shelterin. They protect the chromosomes from damage and play a fundamental role in maintaining genomic stability and integrity. During cell division DNA is replicated to generate new cells. In somatic cells, due to incomplete DNA replication mechanism, telomeres gradually shorten with age until they reach a critical length, leading to cell cycle arrest, cellular senescence, and progressively cell death [ 1 ]. However, in germ cells, granulosa cells, early embryos, stem cells, and various types of cancerous cells, the enzyme telomerase counteracts telomere shortening, helping in the maintenance of genetic information [ 2 ]. Telomere length (TL) has been recognized as a powerful biomarker of biological aging and age-related diseases [ 3 ]. Since biological aging is strongly associated with reproductive aging, extensive research has focused on the possible implication of telomeres in human reproduction [ 4 , 5 ]. In the last decades, people decide to have offspring at advanced age, mostly due to increased life expectancy, late marriages, shifting of personal priorities towards building a career rather than a family, widespread use of contraception, and other factors [ 6 ]. The combined increased average maternal and paternal age has a negative impact on fertility and reproductive capacity. Thus, many couples who face difficulties in conception turn to assisted reproductive technology (ART) treatment in order to have a baby, with in vitro fertilizati on (IVF) being the most common form [ 6 ]. IVF is a technique in which oocytes retrieved after ovarian stimulation are fertilized by sperm in a petri dish, outside the human body, and then fertilized embryos are transferred into the uterus at the cleavage or the blastocyst stage. The number of fertilized embryos transferred depend on the embryo stage and quality, maternal age, and patient preference [ 7 ]. Several factors have been associated with IVF outcome, including parental characteristics (age, gamete quality, infertility, pathologies), ovarian sensitivity, fertilization rate, embryo quality, number of transferred embryos, endometrial thickness, infertility, lifestyle factors, and culture conditions [ 8 , 9 ]. One of the important stages in the IVF is the evaluation, identification, and transfer of high-quality embryos, in order to increase the probability of implantation and pregnancy outcome [ 8 , 9 ]. Growing evidence links altered telomere biology with female and male infertility and pathologies of the female reproductive system [ 4 , 5 ]. Recently, it has been proposed an emerging role of telomeres on embryological parameters and on IVF [ 4 , 5 ]. The purpose of the present article is to further elaborate on the relationship between telomeres and IVF and to evaluate whether TL could serve as a candidate prognostic biomarker of IVF success.