Panminerva Medica Paternal Age and Assisted Reproductive Technologies: Problem Solver Or Trouble Maker?

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Paternal age and assisted reproductive technologies: problem solver or trouble maker?

Mariana BERTONCELLI TANAKA, Ashok AGARWAL, Sandro Cassiano ESTEVES

Panminerva Medica 2018 Jul 18 DOI: 10.23736/S0031-0808.18.03512-7

Article type: Review Article

Article first published online: July 18, 2018 Manuscript accepted: July 16, 2018 Submission Date: June 28, 2018

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EDIZIONI MINERVA MEDICA

Title: Paternal age and assisted reproductive technologies: problem solver or trouble maker?

Authors and Affiliations: Mariana BERTONCELLI TANAKA1, Ashok AGARWAL2, Sandro C. ESTEVES1,3,4

1Department of Surgery (Division of Urology), University of Campinas (UNICAMP), Campinas, SP, Brazil; 2American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, USA, 3ANDROFERT, Andrology and Human Reproduction Clinic, Campinas, SP, Brazil; 4Faculty of Health, Aarhus University, Denmark.

Corresponding author: Sandro C. Esteves, MD., PhD. ANDROFERT, Av. Dr. Heitor Penteado, 1464 13075-460 Campinas, SP, Brazil Tel. +55(19)3295-8877 Fax. +55(19)3294-6992 E-mail: [email protected] ORCID: http://orcid.org/0000-0002-1313-9680

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ABSTRACT In our society, the number of couples with advanced reproductive age seeking fertility treatment is increasing steadily. While the negative effect of female age on assisted reproductive technology (ART) outcomes is well established, the impact of paternal age needs to be clarified. We reviewed the current literature to determine whether advanced paternal age affects the results of ART and the health of resulting offspring. We found that the published literature is overall supportive of a positive association between advanced paternal age (>40 years) and semen quality deterioration. However, the existing evidence does not corroborate nor discard the influence of advanced paternal age on ART outcomes. Similarly, the effect of paternal age on the health of ART offspring remains equivocal, although data from naturally- conceived children clearly indicates that advanced paternal age increases the frequency of genetic, neurodevelopmental, and psychiatric diseases in the progeny. Noteworthy, the current literature is limited and subjected to bias due to the impact of maternal age as a critical confounder. Health care providers should discuss with concerned couples the available options to counteract the possible negative influence of advanced paternal age on ART outcomes and health of resulting offspring. These include identification and treatment of underlying conditions with potential negative long-term effects on fertility, sperm freezing at a young age, and use of antioxidant supplements for men at risk of excessive oxidative stress. Aged male partner from couples undergoing ART, in particular men of 50 years and older, should consider use of preimplantation genetic testing as a means to detect embryo abnormalities and select euploid embryos for transfer to the uterine cavity.

Keywords: paternal age; assisted reproductive technology; in vitro fertilization; intracytoplasmic sperm injection; male infertility.

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Introduction In our society, social, cultural, and economic factors are the main reasons for delayed parenthood. With increasing age, there is a natural decline in fecundability as well as in the ability to carry a pregnancy to term.1 According to the World Health Organization (WHO), infertility rates vary widely worldwide among couples with advanced reproductive age.2 Also, the percentage of parents who first conceive at advanced reproductive age seems to vary between developed and developing countries.1,3-5 It has been estimated that up to 3% of women and 7% men who conceive naturally, respectively, are older than 40 years.1 Among couples seeking assisted reproductive technology (ART) in the United States, approximately 20% are older than 40 years.6 The impact of female age on ART outcomes has been extensively studied. Pregnancy success following in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) is significantly lower in women of advanced maternal age (e.g., 40 years and older) than younger counterparts.1,7-10 The age-related decline in female fertility is mainly driven by deterioration in oocyte quality.11 Oocyte and embryo aneuploidy rates increase steadily with female age, with over 50% embryos being aneuploid in women aged 35 years or older.12,13 Other contributory factors include alterations in the hormonal environment, endometrial pathologies, and the presence of comorbidities that increase in frequency with aging.7,8 It is, therefore, not surprising that reproductive specialists pay special attention to age when counselling and providing care to infertile women.14-17 On the other hand, the influence of paternal age on fertility outcomes, both natural and assisted, remains unclear. Some studies indicate a positive relationship between increased paternal age and poor ART outcomes.18-21 Deterioration of semen quality and changes in the genetic and epigenetic status of spermatozoa with ageing have been implicated as possible causative factors.19,22-25 By contrast, others have failed to confirm any significant influence of paternal age on the results of ART.22,26,27 Noteworthy, assessing the effect of advanced paternal age in ART outcomes is challenging due to the inherent difficulty in avoid maternal age as a confounding factor. There is a paucity of information about the health of offspring delivered through ART from elderly fathers. The risk of genetic- and epigenetic-related diseases, impaired psychological development, as well as decreased fertility in the resulting offspring are some of the concerns associated with the use of ART in general, and particularly in elderly men.

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Given the widespread use of ART and the trend towards an increased patient age among couples seeking fertility treatment, it is essential to elucidate the impact of paternal age on ART outcomes. This understanding may aid healthcare professionals to provide better counselling and care to the affected population. In this review, we firstly evaluate the influence of male age on semen parameters as well as on sperm genetic and epigenetic profiles. Subsequently, we summarize and discuss the available data on the influence of advanced paternal age on ART outcomes. Lastly, we elaborate on practical solutions for men willing to postpone fatherhood.

Impact of Advanced Paternal Age on Sperm Parameters The studies examining the influence of paternal age on conventional semen parameters have yielded mixed results (Table 1).18,19,28-34 In one report, Frattarelli et al. observed a significant decrease in semen volume and motility in men older than 45 years of age. The authors inclusion criteria were based on female infertility referral, independent of male factor.18 In another study, Bellver et al. reported a significant negative association between male age and sperm concentration, motility, and total progressive motility both in fresh ejaculated semen and after preparation by swim-up, although the authors did not provide an age cutoff above which the quality of semen deteriorated.28 Girsh et al. noticed that ejaculate volume, sperm concentration, total sperm count, sperm morphology, sperm motility, and progressive motility were statistically lower in men older than 50 compared to those younger than 40.19 Along the same lines, Duran et al. showed an inverse correlation between male age and total sperm count, ejaculate volume, and percentage of motile sperm.31 Lastly, Beguería et al. observed that semen volume and motile sperm count decreased significantly with age, despite the unexpected increase in sperm concentration that the authors attributed to a possible diminished seminal vesicle function affecting the sperm output.22 By contrast, Ferreira et al. found no correlation between sperm concentration or sperm motility and paternal age in both normozoospermic and oligozoospermic patients.30 In another study, Nijs et al. observed no association between paternal age and sperm concentration and progressive sperm motility.35 The changes in semen parameters seem to be related to a reduction in the number of Sertoli cells with aging, possibly caused by an impaired testicular blood supply.36 The Leydig cell population also decreases with senescence. This leads to a reduction in testosterone production and consequent increase in luteinizing hormone (LH) secretion, in an attempt to maintain testosterone levels within physiological ranges. The follicle-stimulating hormone

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(FSH) also rises in older men in response to increase in inhibin B levels as a means to regulate testicular spermatogenic activity.36 Besides semen parameters and hormone levels, sperm genetic characteristics have also been investigated as a function of paternal age. A classic example is sperm DNA fragmentation (SDF) that occurs in all ages and is the endpoint of a series of conditions associated with increased oxidative stress. The oxidative-induced damage to sperm DNA usually occurs after the male gamete undergoes the postmeiotic phase.37-44 It has been suggested that an oxidative attack on sperm DNA can result in the formation of oxidative base adducts (e.g. 8-hydroxy- 2’-deoxyguanosine [8OHdG]) and aldehyde adducts (4-hydroxynonenal and 4- hydroxyhexenal) on DNA.24 These compounds are mutagenic or immunogenic, and due to the limited capacity of sperm to repair such damages there might be an increased risk of mutation and epigenetic changes in sperm of old men.24 In fact, a number of studies indicate that SDF not only increases with paternal age but also negatively affects ART outcomes.45-51 In the study by Nijs et al., the authors noticed an overall weak but significant correlation between paternal age and sperm chromatin packaging, thus suggesting a detrimental effect of ageing on sperm chromatin integrity. This correlation, however, was not confirmed when the patient population was split into age groups, namely, <34, 35-39, and >40 years-old.35 García-Ferreyra et al. found that men older than 50 years had significantly higher percentages of sperm with fragmented DNA.52 In their study, blastocyst formation rate was significantly lower in this age category, although the percentages of good quality blastocysts were not associated with advancing paternal age. Sperm telomere length (STL) was also shown to be associated with paternal age. Telomeres protect chromosome integrity and have an effect on pairing of the homologous chromosomes and movement of chromosomes during cell division. In one study, Yang et al. demonstrated that STL was positively associated with embryo quality, but not to pregnancy rates.53 While shortening of telomeres causes an arrest of the cell division cycle and induce cellular senescence or apoptosis, in their study STL was longer in older men compared to younger ones. The exact mechanism of longer STL in sperm of aging men is not clear, but there are two possible but not mutually exclusive explanations. The first one suggests that high expression of testicular telomerase might lead to extension of telomeres in sperm chromosomal ends. The second explanation is selective loss of spermatogonial stem cells with shorter telomere length. In line with this hypothesis, stem cells with longer telomeres might acquire mutations that would enable an increased cell division rate similar to pre-neoplastic

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somatic mutation, a process known as “selfish spermatogonial selection”.54 Regardless of the mechanisms involved in STL, there seem to be an influence of telomere length in wound healing, infection resistance, and vascular maintenance, thus suggesting a possible adaptive intergenerational signaling mechanism transmitted by fathers with advanced age to their offspring.54 Sperm aneuploidy rates do not seem to be influenced by paternal age. In a study evaluating numeric chromosomal abnormalities in sperm of semen donors, no differences were observed in older men (aged 60–74, mean age 66.4 years) when compared to younger counterparts (aged 23–37, mean age 27.8 years).55 In other study comparing sperm aneuploidy rates between infertile men with oligoasthenoteratozoospermia (OAT) and fertile men, Andreescu et al. did not find any differences in aneuploidy rates with increasing paternal age.56 The authors showed, however, that sperm from OAT patients exhibited higher rates of aneuploidy than fertile men, with increased nullisomy or disomy of chromosomes 13, 18, 21, X, and Y. By contrast, several studies indicate that chromosomal deletions and duplications as well as balanced translocations increase in sperm of aged men (reviewed by Yatsenko & Turek57 and Gunes et al.58). Apparently, the continuous division of male germ cell line during the entire reproductive lifespan and the oxidative stress-induced sperm DNA damage increase the frequency of mutations in sperm of older men.24 These mutations can be de novo or a result of exogenous factors. Accumulation of chromosomal injuries, recombination errors, and impairment of DNA repair capacity during spermatogenesis, spermiogenesis, and sperm transit through the reproductive tract might explain the increased mutation load in sperm of these men.24,57,58 Base substitutions and DNA repeat extension mutations are the most frequent paternal mutations, both of which increase with advanced paternal age.61 The former leads to mismatching of nucleotides during mitotic cell divisions whereas the latter is associated with replication errors during proliferative stages of spermatogenesis. With ageing, these mutations can accumulate and might increase the risk of disorders linked to paternal age in offspring, such as autosomal dominant disorders caused by mutations in critical genes (e.g., FGFR2, FGFR3, HRAS, PTPN11, RET), including Apert, Crouzon, Pfeiffer, Muenke syndromes, achondroplasia, Costello and Noonan syndromes, and multiple endocrine neoplasia type 2A and type 2B.58,59 Epigenetic changes, which are not related to genetic mutation but rather to changes in gene expression, have also been studied in elderly men. Hyper- and hypo- DNA methylation,

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both of which can change the activity of a DNA segment without changing its sequence, is one of the mechanisms implicated in epigenetic changes.49,50 In one study, Day et al. showed that the pattern of sperm methylation in older men is different from that of their own somatic cells and from their younger counterparts.60 The changes in DNA methylation with ageing might increase the risk of developing specific neurodevelopmental disorders in the resulting offspring.61 Moreover, methylation changes in the promoter regions of genes containing CpG islands in sperm from ageing men might alter the function of genes associated with schizophrenia, bipolar disorders, and autism, thus increasing the risk of these disorders in children of elderly fathers.24,58,60,61 Collectively, the current evidence is overall supportive - albeit not unequivocally - of a negative effect of advanced paternal age on semen parameters and sperm genetic and epigenetics status, with possible preconceptional and post-conceptional implications to both natural fertility as well as ART outcomes.

Impact of Advanced Paternal Age on ART Outcomes

Methodological considerations A number of confounders should be accounted for when evaluating the influence of paternal age on ART results. First, the fertility status of the male partner is an essential element to consider, as it has been shown that the severity of the male factor influences ART outcomes, independently of male age.62-70 Second, external influences, such as abuse of alcohol, smoking, use of substances and medications with potential gonadotoxic effect, obesity and other comorbidities should also be accounted for, as they might impact ART results.62 Third, maternal age as a confounding factor should also be considered, as the age of the women has a major role on ART outcomes. Indeed, the “ideal” model to study solely the effect of paternal age on ART outcomes would be the use of the patients’ own sperm in association with donated oocytes from fertile women. However, even this optimal model is not free of bias as other influences, such as recipient’s endometrial environment, might also play an important role in pregnancy success and perinatal outcomes.71,72 Furthermore, most couples seeking ART have the possibility of using their own gametes, thus creating a need to understand the influence of male age using real world data. In this setting, however, it is well-established that maternal age plays the major role on the likelihood of establishing pregnancy, thus casting a shadow over other possible critical factors, including paternal age.

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Lastly, and to our opinion the critical factor to be weighted when evaluating ART outcomes in ageing males, is the health of resulting offspring. If on the one hand use of ART may overcome infertility issues of ageing couples and allow them to conceive, on the other hand, there might be long-term adverse consequences to the offspring that should be carefully evaluated (Figure 1).73,74 Hence, research should focus not only on periconceptional effects of advanced paternal age but also on short- and long-term postnatal effects to the population of children conceived through ART.

Effect of Advanced Paternal Age on ART outcomes Unlike the well-documented negative effect of maternal age on ART outcomes, data concerning paternal age is relatively limited and results are conflicting (Table 2).

Fertilization rates A few studies report significant decreases in fertilization rate in elderly male groups using an egg donation model. The decline in sperm fertilizing ability seems to start at the age between 45 and 50 years.20,31,75 In the study by Luna et al., after controlling for recipients’ age, the authors observed a significant decrease in fertilization rates particularly among men older than 50 years when IVF was the method of fertilization.20 Similarly, Kaarouch et al. reported lower fertilization rates in men older than 40 years than in their younger counterparts.76 However, the negative effect of paternal age on fertilization rates by ICSI was not observed. Similarly, Tiegs et al. found a significant decrease in fertilization rates with increasing paternal age after controlling for oocyte age.75 Furthermore, Duran et al. showed a significant decrease in fertilization rates related to paternal age in the ICSI subgroup, when comparing males aged between 25 to 38 years to those older than 50 years (p=0.008).31

Embryo quality Four studies assessed the effect of paternal age on embryo quality. Luna et al. observed that both the number of embryos with more than 7 cells on day 3 and blastulation rates significantly decreased as paternal age progressed.20 Girsh et al., using an egg donation model in an ICSI exclusive setting, divided couples in pregnant and non-pregnant, based on the results of ß-hCG.19 They found that men were significantly younger in the pregnant group, with an average age of 43.2±8.1 years, compared with those in the non-pregnant group (average age of 46.8±7.8 years; p=0.003). They observed that the percentage of good quality embryos on day 3 was significantly lower in the non-pregnant group (26%), as compared with the pregnant group (34%; p=0.01). Frattarelli et al., also using an egg donor model, observed

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a significant decrease in blastocyst formation among men older than 55 years.18 In their study, the age cut-off from which blastulation decreased was 50 years. Men younger than 50 had 40.7% of their embryos reaching the blastocyst stage by day 5, whereas men >50 years-old had 34.2% of their embryos reaching this stage of embryo development (Relative risk [RR]=1.19, 95% CI: 1.09–1.30; p<0.01).18 Lastly, Chapuis et al. reported a decrease in blastocyst formation rate among couples with male partners older than 50 years undergoing IVF (p=0.0255).77 These results, however, were not observed among couples subjected to ICSI.

Pregnancy Rates Campos et al. observed a negative influence of advanced paternal age on clinical pregnancy in an egg donation setting, but the authors acknowledged that maternal age was a possible confounder.29 When one of the parents was younger than 39 years-old, the negative association between age and pregnancy lost significance. In another study, Wu et al. observed a decrease in clinical pregnancy rates in men older than 36 years compared to those younger than 32 years among couples whose maternal age was between 30 and 34 years.79 Similarly, Ferreira et al. evaluated ICSI outcomes as a function of paternal age in normozoospermic and oligozoospermic men.30 After controlling for maternal age, number of oocytes retrieved, sperm concentration, and fertilization rate, the authors observed a decrease in implantation and pregnancy rates only among older fathers with oligozoospermia. The authors concluded that the chances of pregnancy success decrease 5% for each year of increased paternal age in oligozoospermic patients.30 In a ICSI study involving men with azoospermia, Park et al. reported that clinical pregnancy rates were decreased among couples where the male partners were older than 46 years, independent of the cause of azoospermia.78 A positive association between increased pregnancy loss and advanced paternal age was reported by Frattarelli et al.18 The authors found that the rates of pregnancy loss differed significantly in men of younger (41.5%) and older than 50 years (24.4%; RR 0.61, 95% CI 0.45–0.84; p<0.01). Other studies mentioned a trend towards increased pregnancy loss rates in older fathers, but the results did not reach statistical significance.20,77 A few studies investigated the influence of paternal age on live birth rates (LBR).18,21,80 Notably, McPherson et al. reported a decrease in LBR among couples with advanced maternal (>35) and paternal (>40) age.80 The authors hypothesized that the negative effect of age on SDF, the oocyte's cytoplasm DNA repair mechanisms, and endometrial receptivity might explain these poor reproductive outcomes among aged couples. However, the authors of this

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study cautioned that the negative effect of paternal age on ART outcomes was far less remarkable than the influence of maternal age. By contrast, other groups showed no effect of paternal age on ART outcomes. In one study, Bellver et al. observed no significant difference in pregnancy rates and miscarriage rates among different paternal age groups.28 In the egg donor subgroup, the authors identified an increase in embryo fragmentation associated with paternal age, but the correlation was very low, thus questioning the clinical significance of their findings. In another study involving 278 couples, Nijs et al. observed no difference in fertilization rates, pregnancy rates, and LBRs when maternal age was controlled.35 In this study, however, men with severe oligoasthenoteratozoospermia (OAT), those who used testicular sperm for ICSI, and cases involving pre-implantational genetic testing were excluded. It is worth noting that most studies that reported a lack of influence of paternal age on ART outcomes included small patient cohorts and fewer men older than 40 years.28,35,81,82 Other studies, however, included large cohorts and took into account variables such as maternal age and the number of embryos transferred per cycle. For instance, Meijerink et al. evaluated the influence of paternal age among 7051 couples on their first ART attempt.83 The authors also assessed the role of paternal age according to the source of sperm used for ICSI, i.e., ejaculated sperm and sperm retrieved by PESA, MESA, and TESE. In this large cohort study, no association was found between paternal age and ongoing pregnancy rates by ART, nor did the sperm source influence pregnancy results. Likewise, Whitcomb et al.26 did not find a detrimental effect of paternal age in an egg donor model involving a cohort of 1083 couples after adjusting for female age.26 Lastly, Begueria et al. in an egg donation model with ICSI as the solely fertilization method, found that male age was not associated with any pregnancy outcome, namely, biochemical pregnancy rate (RR: 1.0; 95% CI 0.96–1.05), miscarriage rate (RR: 1.06; 95% CI 0.94–1.03), ongoing pregnancy rate (RR: 0.98; 95% CI 0.94–1.033), and LBR (RR: 0.98; 95% CI 0.94–1.03).22 Similar findings were reported by Ghuman et al. who evaluated the effect of advanced paternal age (40 to 45 years) in a sperm donation model.84 They found no correlation between paternal age and miscarriage or LBR after controlling for female age, treatment modality, and the effect of previous treatment cycles. Notwithstanding these findings, the authors cautioned that generalization of their results might not be possible, because sperm were provided by a cohort of donors, whereas most men seeking ART have altered sperm parameters.

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In conclusion, despite the existence of limited evidence suggesting a negative effect of advanced paternal age on ART outcomes, this association remains inconclusive. In all studies, however, it is clear that maternal age is the most prominent factor determining pregnancy success following IVF and ICSI.

How old is old? Despite the fair number of studies suggesting a negative influence of paternal age on ART outcomes, it is not yet clear whether there is a precise age cutoff at which risk develops nor if the risk does gradually increase as the man ages. The diversity of study protocols and populations, adds to the various outcome measures investigated, make it difficult to qualitatively compare and meta-analyze the available data. While some studies show a linear correlation between paternal age and ART outcomes,21,80 others show a significant influence only from a specific age onwards.18,20,29,30,79,85 By all means, in studies indicating that paternal age has a negative effect on ART outcomes, it seems that the risk is higher among men of 40 years and older. In fact, according to the clinical practice guidelines issued by the Society of Obstetricians and Gynecologists of Canada (SOGC)86 and the American College of Obstetricians and Gynecologists (ACOG)87, men of 40 years and older undergoing ART might be considered “old fathers” and counselled accordingly about (i) the risks of delaying parenthood further, (ii) the potential decrease in ART outcomes, and (iii) the increased health risks for the resulting offspring.

Influence of paternal age on the health of resulting offspring An increasing body of evidence indicates that postponing fatherhood can negatively influence the health of resulting offspring. Genetic defects related to faulty sperm quality control leading to single gene mutations and epigenetic alterations in several genetic pathways have been implicated as root causes.88 As previously discussed, advanced paternal age might lead to an accumulation of damage to sperm DNA and mismatch repair, which seems to be related to deficient sperm quality control during spermatogenesis. This in turn is translated by increased sperm DNA fragmentation, single gene mutations, and abnormalities in sperm chromosomes (reviewed by Yatsenko & Turek, and Aitken).24,57 Epigenetic alterations in several genetic pathways might also be implicated as root causes of other diseases, including autism and schizophrenia. Most of these data, however, derives from studies involving naturally conceived children, and at present it is uncertain to what extend these results can be extrapolated to the population undergoing ART.

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Reichenberg et al. observed that naturally conceived children from fathers aged 40 years or older were 5.57 times more likely to have an autism spectrum disorder.73 This association was present in those with no family history of the disorder, thus indicating paternal age as an independent risk factor for autism. In another study involving naturally conceived children, Sipos et al. identified a hazard ratio of 1.47 (95% CI 1.23 to 1.76) for schizophrenia for each 10-year increase in paternal age.74 Other diseases have been also associated with advanced paternal age, such as bipolar disorder,89 abnormal neurocognitive development,90 breast cancer in women,91 and birth defects.92 Notably, a trend for increased birth defects were observed in both extremes of age; the authors speculated that birth defects in offspring from very young fathers (<20 years) might be due to increased maternal exposure to external agents or unplanned pregnancy, possibly leading to folate deficiency.92 By contrast, there is a paucity of ART data concerning the health of offspring from older fathers (Table 3). In one study, Stern et al. found that the risks for prematurity, low birthweight, and small for gestational age were not influenced by advanced paternal age.93 In another study, Wu et al. showed no effect on preterm births (OR=1.06; 95% CI 0.89–1.25).27 Likewise, Hurley et al., in a subgroup analysis among couples undergoing ART, did not observe an association between advanced paternal age and increased rates of preeclampsia, preterm birth, fetal growth restriction, congenital anomaly, genetic disorder, or neonatal intensive care unit admission.94 From these limited data, it seems that advanced paternal age has minimal influence on short-term ART perinatal outcomes. However, a call for active surveillance is required as there is convincing evidence indicating that advanced paternal age has a negative effect on the fate of naturally-conceived children. Moreover, there is a lack of published ART data regarding epigenetic inheritance, chromosomal abnormalities, as well as psychological and neurodevelopmental disorders of offspring resulting from old fathers (Figure 1).

Possible solutions for men willing to postpone fatherhood Despite the growing literature regarding ART in older men, many questions remain unanswered. A counselling guideline with clear recommendations for men with advanced age seeking fertility has not been developed yet. From the practical point of view, counselling is based on individual patient’s physical condition, presence of comorbidities, as well as social and cultural backgrounds. Oral antioxidants have been widely utilized as a means to alleviate the oxidative stress- induced sperm damage in the male infertility population, with mixed results.95,96 This strategy

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might be also considered in older men, whose sperm are more prone to oxidative damage. However, likewise in the general male infertility population, it remains to be determined what would be the optimal combination of antioxidants, dosage and duration. While use of oral antioxidant supplements could be useful to improve male reproductive health in older men, there are potential risks that the long-term use might cause a paradoxical antioxidant inhibition of essential and beneficial ROS activities to male reproductive biology.97 Other aspect when counselling prospective fathers is to discuss the pros and cons of delayed parenthood. For instance, sperm freezing might be offered to young men considering postponing fatherhood as an intervention aiming at counteracting the effect of age on fertility. Nevertheless, important issues should be weighted when considering elective sperm cryopreservation as thawed sperm will need to be used in association with assisted treatment modalities. If on the one hand the use of a younger patient’s semen would theoretically decrease the risks of Down syndrome, schizophrenia, and autism spectrum disorders, on the other hand the quality of sperm and even DNA integrity could be harmed during the freeze- thaw process.98 Besides these risks, other ethical, legal, and financial concerns should be raised and thoroughly evaluated when counselling prospective fathers for sperm cryopreservation. Unlike preserving sperm for patients with nonobstructive azoospermia (NOA) or undergoing chemotherapy, there are no specific guidelines that address sperm freezing aiming only to postpone paternity,99 so pros and cons should be discussed with each patient individually. Counselling older men who will undergo ART should include discussion about use of preimplantation genetic testing (PGT). PGT for aneuploidy screening (PGT-A) has been widely utilized as a means to detect embryo abnormalities and select euploid embryos for transfer to the uterine cavity, in particular, among women with advanced age and those suffering from recurrent pregnancy loss. The method is also used to test for specific genetic conditions including monogenic and single gene defects (PGT-M) and chromosomal structural rearrangements (PGT-SR). With the use of PGT, implantation rates in older women may increase by 18% compared to unscreened ICSI embryos.100,101 Despite the potential use of PGT-A for advanced paternal age, there is paucity of published data concerning the effect of paternal age on the embryo euploidy status. In a 2012 study, Braga et al. evaluated the influence of paternal age on aneuploidy rates by FISH on day 3 embryos and found no association.102 By contrast, Garcia-Ferreyra et al.52 showed a significant increase in aneuploidy rates in embryos of men older than 50 years, in the context of egg donation.52 The authors of this study suggested that embryo genetic screening might be

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performed in men older than 50 years as a means to reduce miscarriage and improve ART clinical outcomes. Although PGT may improve implantation rates in older parents, it can also pose risks associated to the technical aspects of embryo biopsy and cryopreservation. However, recent development in trophectoderm biopsy technique and embryo vitrification have minimized these risks significantly. Yet, it is worth mentioning that PGT-A adds substantial expenses to the already costly ART. Lastly, counseling infertile patients, including the ageing subpopulation, about the risks of smoking, gonadotoxin exposure, and alcohol abuse is equally critical as all of these conditions might impact fertility.103,104 Although identification and treatment of underlying conditions associated with infertility is often neglected among couples undergoing ART, these aspects should be integral elements of care for men seeking fertility regardless of age group as they might optimize ART outcomes.39,40,46,49 Taken together, use of antioxidants, adoption of a healthy lifestyle, treatment of underlying conditions associated with infertility, and sperm banking are valid alternatives for younger considering postponing fatherhood (Figure 2). For those undergoing ART at later reproductive age, in particular if older than 50 years, the use of PGT-A should be considered.

Conclusions

Men at the age of 40 years or greater are at increased risk of having genetic mutations, DNA fragmentation, and epigenetic changes in sperm. Moreover, there is an overall deterioration in conventional semen parameters with ageing. Although advanced paternal age seems to have a minor influence on ART outcomes when compared to maternal age, further studies are needed to elucidate this association as the current literature is limited and subjected to bias owed to the impact of maternal age as a critical confounder. Whether the risk of genetic, neurodevelopmental, and psychiatric diseases is increased among ART children from older fathers is unclear, the evidence from non-ART population clearly indicates that the risk of these diseases is higher in children from older fathers than in children from younger fathers. All efforts should be made to properly counsel men willing to postponing fatherhood about the possible decreased success in ART and increased risks of diseases in offspring when sperm from old fathers are used for fertilization. Options for prospective fathers postponing parenthood include identification and treatment of underlying conditions with potential negative long-term effects on fertility, sperm freezing at a young age, and use of antioxidant

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agents for men at risk of excessive oxidative stress. Aged men undergoing ART, in particular those of 50 years and older, should consider use of pre-implatation genetic testing as a means to detect embryo abnormalities and select euploid embryos for transfer to the uterine cavity.

Review Criteria An extensive search of studies examining the influence of paternal age on ART outcomes was performed using PubMed and MEDLINE. The start date for the search was January 2008, and the end date was March 2018. The overall strategy for study identification and data extraction was based on the following keywords: “paternal age”, “male age factor”, “masculine age factor”, “male ageing”, “paternal ageing”, “reproductive techniques”, “assisted”, “ICSI”, “in vitro fertilization” “sperm injections”, “intracytoplasmic”, “IVF”, with the filters “humans” and “English language.” Data that were solely published in conference or meeting proceedings, websites or books were not included. Citations dated outside the search dates were only included if provided conceptual content.

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16. Alviggi C, Andersen CY, Buehler K, Conforti A, De Placido G, Esteves SC, et al. A new more detailed stratification of low responders to ovarian stimulation: from a poor ovarian response to a low prognosis concept. Fertil Steril 2016;105:1452-3. 17. Humaidan P, Alviggi C, Fischer R, Esteves SC. The novel POSEIDON stratification of ‘Low prognosis patients in Assisted Reproductive Technology’ and its proposed marker of successful outcome. F1000Res 2016;5:2911. 18. Frattarelli JL, Miller KA, Miller BT, Elkind-Hirsch K, Scott RT Jr. Male age negatively impacts embryo development and reproductive outcome in donor oocyte assisted reproductive technology cycles. Fertil Steril. 2008;90:97-103. 19. Girsh E, Katz N, Genkin L, Girtler O, Bocker J, Bezdin S, et al. Male age influences oocyte-donor program results. J Assist Reprod Genet 2008;25:137-43. 20. Luna M, Finkler E, Barritt J, Bar-Chama N, Sandler B, Copperman AB, et al. Paternal age and assisted reproductive technology outcome in ovum recipients. Fertil Steril 2009;92:1772- 5. 21. Robertshaw I, Khoury J, Abdallah ME, Warikoo P, Hofmann GE. The effect of paternal age on outcome in assisted reproductive technology using the ovum donation model. Reprod Sci 2014;21:590-3. 22. Beguería R, García D, Obradors A, Poisot F, Vassena R, Vernaeve V. Paternal age and assisted reproductive outcomes in ICSI donor oocytes: is there an effect of older fathers? Hum Reprod 2014;29:2114-22. 23. Elhanbly S, El-Saied MA, Fawzy M, El-Refaeey A, Mostafa T. Relationship of paternal age with outcome of percutaneous epididymal sperm aspiration-intracytoplasmic sperm injection, in cases of congenital bilateral absence of the vas deferens. Fertil Steril 2015;104:602-6. 24. Aitken RJ. Not every sperm is sacred; a perspective on male infertility. Mol Hum Reprod 2018 ahead of print, doi: 10.1093/molehr/gay010. 25. Jenkins TG, Aston KI, Pflueger C, Cairns BR, Carrell DT. Age-associated sperm DNA methylation alterations: possible implications in offspring disease susceptibility. PLoS Genet 2014;10:e1004458. 26. Whitcomb BW, Turzanski-Fortner R, Richter KS, Kipersztok S, Stillman RJ, Levy MJ, et al. Contribution of male age to outcomes in assisted reproductive technologies. Fertil Steril 2011;95:147-51.

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63. Agarwal A, Cho CL, Esteves SC. Should we evaluate and treat sperm DNA fragmentation? Curr Opin Obstet Gynecol. 2016;28:164-71. 64. Esteves SC, Roque M, Agarwal A. Outcome of assisted reproductive technology in men with treated and untreated varicocele: systematic review and meta-analysis. Asian J Androl 2016;18:254-8. 65. Esteves SC, Sánchez-Martín F, Sánchez-Martín P, Schneider DT, Gosálvez J. Comparison of reproductive outcome in oligozoospermic men with high sperm DNA fragmentation undergoing intracytoplasmic sperm injection with ejaculated and testicular sperm. Fertil Steril 2015;104:1398-405. 66. Esteves SC. Clinical management of infertile men with nonobstructive azoospermia. Asian J Androl 2015;17:459-70. 67. Esteves SC, Prudencio C, Seol B, Verza S, Knoedler C, Agarwal A. Comparison of sperm retrieval and reproductive outcome in azoospermic men with testicular failure and obstructive azoospermia treated for infertility. Asian J Androl 2014;16:602-6. 68. Esteves SC, Agarwal A. Reproductive outcomes, including neonatal data, following sperm injection in men with obstructive and nonobstructive azoospermia: case series and systematic review. Clinics (Sao Paulo). 2013;68 Suppl 1:141-50. 69. Esteves SC, Oliveira FV, Bertolla RP. Clinical outcome of intracytoplasmic sperm injection in infertile men with treated and untreated clinical varicocele. J Urol 2010;184:1442- 6. 70. Verza S Jr, Esteves SC. Sperm defect severity rather than sperm Source is associated with lower fertilization rates after intracytoplasmic sperm injection. Int Braz J Urol 2008;34:49-56. 71. Fox C, Morin S, Jeong JW, Scott RT Jr, Lessey BA. Local and systemic factors and implantation: what is the evidence? Fertil Steril 2016;105:873-84. 72. Moreno I, Franasiak JM. Endometrial microbiota-new player in town. Fertil Steril 2017;108:32-9. 73. Reichenberg A, Gross R, Weiser M, Bresnahan M, Silverman J et al. Advancing paternal age and autism. Arch Gen Psychiatry 2006; 63:1026–32. 74. Sipos A, Rasmussen F, Harrison G, Tynelius P, Lewis G et al. Paternal age and schizophrenia: a population based cohort study. BMJ 2004;329:1070. 75. Tiegs AW, Sachdev NM, Grifo JA, McCulloh DH, Licciardi F. Paternal age is not associated with pregnancy outcomes after single thawed euploid blastocyst transfer. Reprod Sci 2017;24:1319-24.

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76. Kaarouch I, Bouamoud N, Madkour A, Louanjli N, Saadani B, Assou S, et al. Paternal age: Negative impact on sperm genome decays and IVF outcomes after 40 years. Mol Reprod Dev 2018; 85:271-280. 77. Chapuis A, Gala A, Ferrières-Hoa A, Mullet T, Bringer-Deutsch S, Vintejoux E, et al. Sperm quality and paternal age: effect on blastocyst formation and pregnancy rates. Basic Clin Androl 2017;27:2. 78. Park YS, Lee SH, Lim CK, Choi HW, An JH, Park CW, et al. Paternal age as an independent factor does not affect embryo quality and pregnancy outcomes of testicular sperm extraction-intracytoplasmic sperm injection in azoospermia. Andrologia 2018;50(2). doi: 10.1111/and.12864. Epub 2017 Jul 13. 79. Wu Y, Kang X, Zheng H, Liu H, Liu J. Effect of Paternal Age on Reproductive Outcomes of In Vitro Fertilization. PLoS One 2015;10:e0135734. 80. McPherson NO, Zander-Fox D, Vincent AD, Lane M. Combined advanced parental age has an additive negative effect on live birth rates-data from 4057 first IVF/ICSI cycles. J Assist Reprod Genet 2018;35:279-87. 81. Tsai YR, Lan KC, Kung FT, Lin PY, Chiang HJ, Lin YJ, Huang FJ. The effect of advanced paternal age on the outcomes of assisted reproductive techniques among patients with azoospermia using cryopreserved testicular spermatozoa. Taiwan J Obstet Gynecol 2013;52:351-5. 82. Gu L, Zhang H, Yin L, Bu Z, Zhu G. Effect of male age on the outcome of in vitro fertilization: oocyte donation as a model. J Assist Reprod Genet 2012;29:331-4. 83. Meijerink AM, Ramos L, Fleischer K, Veltman JA, Hendriks JC, Braat DD. Influence of paternal age on ongoing pregnancy rate at eight weeks' gestation in assisted reproduction. Reprod Biomed Online 2016;32:96-103. 84. Ghuman NK, Mair E, Pearce K, Choudhary M. Does age of the sperm donor influence live birth outcome in assisted reproduction? Hum Reprod 2016;31:582-90. 85. Ramasamy R, Trivedi NN, Reifsnyder JE, Palermo GD, Rosenwaks Z, Schlegel PN. Age does not adversely affect sperm retrieval in men undergoing microdissection testicular sperm extraction. Fertil Steril 2014;101:653-5. 86. Liu K, Case A. Advanced reproductive age and fertility. J Obstet Gynaecol Can 2011;33:1165–75. 87. Toriello HV1, Meck JM; Professional Practice and Guidelines Committee. Statement on guidance for genetic counseling in advanced paternal age. Genet Med 2008;10:457-60.

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88. Conti SL, Eisenberg ML. Paternal aging and increased risk of congenital disease, psychiatric disorders, and cancer. Asian J Androl 2016;18:420-4. 89. Frans EM, Sandin S, Reichenberg A, Lichtenstein P, Lå ngström N, et al. Advancing paternal age and bipolar disorder. Arch Gen Psychiatry 2008; 65:1034–40. 90. Saha S, Barnett AG, Foldi C, Burne TH, Eyles DW, et al. Advanced paternal age is associated with impaired neurocognitive outcomes during infancy and childhood. PLoS Med 2009;6:e40. 91. Choi JY, Lee KM, Park SK, Noh DY, Ahn SH, et al. Association of paternal age at birth and the risk of breast cancer in offspring: a case control study. BMC Cancer 2005;5:143. 92. Yang Q, Wen SW, Leader A, Chen XK, Lipson J, et al. Paternal age and birth defects: how strong is the association? Hum Reprod 2007; 22: 696–701. 93. Stern JE, Luke B, Hornstein MD, Cabral H, Gopal D, Diop H, Kotelchuck M. The effect of father's age in fertile, subfertile, and assisted reproductive technology pregnancies: a population based cohort study. J Assist Reprod Genet 2014;31:1437-44. 94. Hurley EG, DeFranco E. Influence of paternal age on perinatal outcomes. Am J Obstet Gynecol 2017;217:566. 95. Showell MG, Mackenzie-Proctor R, Brown J, Yazdani A, Stankiewicz MT, Hart RJ. Antioxidants for male subfertility. Cochrane Database of Systematic Reviews 2014, Issue 12. Art. No.: CD007411. DOI: 10.1002/14651858.CD007411.pub3 96. Calogero AE, Condorelli RA, Russo GI, La Vignera S. Conservative nonhormonal options for the treatment of male infertility: antibiotics, anti-Inflammatory drugs, and antioxidants. Biomed Res Int 2017;2017:4650182. 97. Leisegang K, Henkel R, Agarwal A. Redox regulation of fertility in aging male and the role of antioxidants: a savior or stressor. Curr Pharm Des 2017;23:4438-50. 98. Donnelly ET, Steele EK, McClure N, Lewis SE. Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Hum Reprod 2001;16:1191–9. 99. Jennings MO, Owen RC, Keefe D, Kim ED. Management and counseling of the male with advanced paternal age. Fertil Steril 2017;107:324-8. 100. Munne S, Sandalinas M, Escudero T, Velilla E, Walmsley R, Sadowy S, et al. Improved implantation after preimplantation genetic diagnosis of aneuploidy. Reprod Biomed Online 2003;7:91–7.

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101. Staessen C, Verpoest W, Donoso P, Haentjens P, Van der Elst J, Liebaers I, et al. Preimplantation genetic screening does not improve delivery rate in women under the age of 36 following single-embryo transfer. Hum Reprod 2008;23:2818–25. 102. Braga DP, Setti AS, Figueira Rde C, Iaconelli A Jr, Borges E Jr. Contributing factors for the incidence of aneuploidy in older patients undergoing intracytoplasmic sperm injection cycles. J Assist Reprod Genet. 2012;29:911-6. 103. Govindarajan M, Mallikarjunan S, Ahmed HSR, Lakshmanan N, Silambuchelvi D, Sujatha M, et al. Retrospective study of factors affecting intrauterine insemination pregnancy outcome: the impact of male habits and working environment. J Hum Reprod Sci 2017;10:114-8. 104. Drobnis EZ, Nangia AK.Male reproductive functions disrupted by pharmacological agents. Adv Exp Med Biol. 2017;1034:13-24.

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Authors’ contributions: MBT participated in the acquisition of data, helped in data interpretation, and drafted the manuscript; AA helped in data interpretation and drafted the manuscript; SCE; designed the study, helped in data interpretation and coordination, and drafted the manuscript. All authors read and approved the final manuscript.

Conflicts of Interest The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

Funding None

Acknowledgement

Professor Ralf Henkel, Department of Medical Biosciences, University of the Western Cape, South Africa, for his critical review of the manuscript.

Legends to Figures Figure 1. Effect of advanced paternal age on semen quality and sperm genetic/epigenetic status and its possible consequences to assisted reproductive technology outcomes. Figure 2. Possible solutions for men willing to postpone fatherhood.

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Table 1. Summary of studies evaluating the effect of paternal age on semen quality and the age cutoff at which the negative effect is observed

Study and Country Number Population Main findings Age Year patients cutoff Frattarelli et USA 1023 Couples seeking No change in sperm 45 al., 2008 ART for female concentration, motility, or infertility reasons. morphology among different Males with and age groups, albeit a decrease in without abnormal semen volume was noted in semen. No older men. Men <45 years had testicular biopsy or significantly higher semen sperm aspiration volume than men >45 years procedures were (P<0.001). Linear regression included analysis revealed a significant decline in semen volume as men aged (r = -0.28, P<0.001). Men aged <45 years had significantly more motile sperm than men >45 years (P<0.05). Linear regression analysis revealed a significant decline in the total number of motile sperm as men aged (r=0.24, P<0.01)

Bellver et Spain 2698 Couples seeking A significant negative NR al., 2008 ART for female association was found between infertility reasons. male age and semen volume, Couples with sperm concentration, motility, severe male factor and total progressive motility in (<5 x 106/ml) were fresh spermatozoa and after excluded swim-up. However, among all variables, only fresh ejaculate progressive motility and ejaculate volume were considered clinically relevant because the Pearson’s correlation coefficients were around or over 0.3. (P<0.05)

Girsh et al., Israel 484 Couples seeking Male age increased as sperm Between 2008 ART for female morphology declined: 40 and 50 infertility reasons. normozoospermic men Males with and (44.8±8.3 years-old), mild without semen teratozoospermic (47.9±7.7 abnormalities. No years-old; P=0.02), moderate testicular biopsy or teratozoospermic (48.4±7.0 sperm aspiration years-old; P=0.04) and severe procedures were teratozoospermic men (51.9±7.3 included years-old; P=0.001). Analysis of sperm using the strict criteria revealed a significantly higher frequency of men with teratozoospermia in the non- pregnant group (28%) than in the pregnant group (11%; P=0.04). Among men with normal morphology, male age

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was higher in the non-pregnant group than in the pregnant group (45.2±8.4 vs. 42.9±8.0, respectively; P=0.05). Semen volume, sperm concentration, total sperm count, sperm morphology, total sperm motility and progressive motility were significantly lower in men aged >50 than in men aged <40 years (P<0.05) Ferreira et Brazil 1024 Normozoospermic No correlation between sperm NR al., 2010 and concentration and paternal age oligozoospermic in both normozoospermic patients (P=0.15; Pearson r=0.055) or oligozoospermic patients (P=0.38; Pearson r=0.048). Sperm motility was also not correlated with paternal age in normozoospermic (P=0.13; Pearson r=-0.057) and oligozoospermic patients (P=0.30; Pearson r=-0.056)

Duran et al., USA 408 Couples seeking Semen volume, sperm motility NR 2010 ART for female and fertilization rates were infertility reasons. inversely correlated with male Males with and age. All of these parameters without semen were adversely and significantly abnormalities. No affected by advanced male age. testicular biopsy or There was a significant inverse sperm aspiration correlation between male age procedures were and total sperm count included (Spearman’s r=0.133, P=0.003), low semen volume (P=0.001, r=0.149), and percentage motile spermatozoa (P<0.001, r=- 0.155)

Nijs et al., Belgium 278 No statistical correlation NR Couples seeking 2011 between the age of the patient ART for male, and sperm concentration, sperm female or mixed progressive motility, and DNA infertility reasons. fragmentation. Although no Extreme OAT cases correlation was observed overall and testicular between male age and sperm spermatozoa morphology, a weak inverse excluded correlation was found for men aged <35 years-old (B=0.191; P=0.027)

Varshini et India 504 Couples seeking TUNEL-positive DNA damaged 40 al., 2011 ART for male sperm was significantly higher infertility reasons in men older than 40 years than in those younger than 40 years (P< 0.001)

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Stone et al., USA 5081 Couples seeking Total sperm numbers in 35 2013 ART for male, ejaculates fall by ~2% per year female or mixed from 34 years-old onwards (P< infertility reasons 0.001). Sperm concentration and morphology were negatively affected in men aged 40 years or older, each diminishing by ~0.8% per year. Total sperm motility and progressive motility declined by ~2% and 0.8% per year, respectively, from age 43 onwards

Alshahrani USA 472 Non-azoospermic No significant differences in 40 et al., 2014 infertile men conventional semen parameters, TAC and ROS levels in the 4 age groups (<30, 31-40, <40, >40). Higher levels of DNA damage were seen in men >40 y when compared with men ≤40 y (P < 0.05) as well as in the overall group (P < 0.01)

Beguería et Spain 4887 Couples seeking Semen volume decreased as NR al., 2014 ART for female men aged, with a mean infertility reasons. reduction of 0.22 ml every 5 Males with and years [95% CI 0.17–0.26] without semen (P<0.001). Sperm concentration abnormalities. No increased with a mean increase testicular biopsy or of 3.1 million sperm per ml sperm aspiration every 5 years of age [95% CI procedures were 1.1–5.2] (P=0.003). The included percentage of motile spermatozoa decreased, with a mean reduction of 1.2% every 5 years of age [95% CI 0.84–1.15] (P<0.001)

Kaarouch et Morocco 204 Couples seeking No significant differences in 40 al., 2018 ART for male conventional semen parameters infertility reasons as a function of paternal age. Higher rates of sperm DNA fragmentation (41% vs. 14%; P=0.01), sperm DNA decondensation (43% vs. 25%; P=0.01), and sperm aneuploidy (23% vs. 4%; P=0.04) in men older than 40 years than in those <40 years NR: not reported; CI confidence interval; TAC: Total antioxidant capacity; ROS: Reactive oxygen species

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Table 2. Summary of studies evaluating the effect of paternal age on ART outcomes Use of egg Miscarriage Age Author and Number Insemination Fertilization Embryo Pregnancy Country donation and Live birth rates cutoff Year (cycles) method rates development rates model stillbirth (years) Gu et al., 2012 China 103 IVF Yes No effect NR No effect No effect No effect NA There was a 26% lower odds of achieving a LB Progressive with each 5-year increase in increase in paternal age rates of (odds ratio 0.73, 95% Robertshaw et pregnancy USA 237 IVF Yes NR NR NR CI: 0.6-0.91, NR al., 2014 loss with P<0.01), after increased controlling for embryo paternal age grade, number of (P<0.01) embryos transferred, and donor age Decreased in men >36 y compared to Wu et al., China 9991 IVF No No effect No effect men <32 y No effect No effect 36 2015 and women 30-34 y (P<0.05) Good-quality Decreased day 3 with Girsh et al., decreased advanced Israel 484 ICSI Yes No effect NR NR 40-50 2008 with advanced paternal age paternal age (P-value not (P=0.01) given) Decreased by Ferreira et al., 5% for each Brazil 1024 ICSI No NR No effect No effect NR NR 2010 year of paternal age

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among oligozoosper mic (P=0.017) Tsai et al., Taiwan 184 ICSI No No effect No effect No effect No effect No effect NA 2013 Decreased with Ramasamy et USA 1046 ICSI No NR NR advanced NR NR 50 al., 2014 paternal age (P=0.01) Beguería et Spain 4887 ICSI Yes No effect No effect No effect No effect No effect NA al., 2014 Reduced Increased blastocyst miscarriage Decreased with Frattarelli et formation USA 1023 IVF and ICSI No No effect No effect with advanced advanced paternal age 55 al., 2008 with advanced paternal age (P<0.01) paternal age (P<0.05) (P<0.05)

Bellver et al., Spain 2698 IVF and ICSI Yes No effect No effect No effect No effect NR NA 2008

Decreased with Campos et al., Spain 915 IVF and ICSI Yes No effect No effect advanced No effect NR 39 2008 paternal age (P=0.01) Decreased Reduced with blastocyst 40* Luna et al., advanced formation USA 672 IVF and ICSI Yes No effect No effect NR and 2009 paternal with advanced 60‡ age* paternal (P<0.05) age* (P<0.05)

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Decreased with Duran et al., USA 519 IVF and ICSI Yes advanced No effect No effect No effect No effect 50 2010 paternal age (P=0.008)

Ghuman et al., UK 7104 IVF and ICSI No NR NR No effect No effect NR NA 2016

Meijerink et Netherla 7051 IVF and ICSI No No effect No effect No effect NR NR NA al., 2016 nds

An approximate 10% further decrease in pregnancy and LBRs was recorded in women McPherson et aged 35 years when Australia 2215 IVF and ICSI No No effect NR See LBR NR 40 al., 2017 their partner was >40 years compared with women aged 35 years with a partner <30 years (P=0.04) Decreased with Tiegs et al., USA 573 IVF and ICSI Yes advanced No effect No effect No effect No effect 45 2017 paternal age (P=0.04) Reduced No effect on blastocyst IVF formation in conventional Decreased Chapuis et al., France 2491 IVF and ICSI No No effect IVF with with No effect — 50 2017 advanced advanced paternal age paternal age years in ICSI (P=0.02) (P=0.01)

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Decreased in Park et al., men >46 Decreased in men >46 Korea 452 ICSI No No effect No effect NR 46 2017 years-old years-old (P<0.05) (P<0.05)

Decreased Decreased Decreased Increased Kaarouch et with with with advanced with advanced al., Morocco 83 IVF No advanced advanced NR 40 paternal age paternal age 2018 paternal age paternal age (P=0.03) (P=0.04) (P=0.02) (P=0.04)

NR: not reported; NA: not applicable; IVF: in vitro fertilization; ICSI: intracytoplasmic sperm injection; LBR: live birth rate; LB: live birth

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Table 3. Summary of studies evaluating the impact of paternal age on the health of ART- conceived offspring Study Country Study Number of Main findings subjects

Stern et USA Population-based 9,092 Risks for prematurity, low birthweight, and al., 2014 retrospective cohort small for gestational-age not affected by advanced paternal age (adjusted OR estimate 95% confidence intervals crossing the null value of 1)

Wu et al., China Retrospective 1249 No effect on preterm births (OR 1.06; 95% 2016 cohort CI 0.898–1.254) No statistical differences in the rates of birth defects between the three age groups (<30 years: 0.3%; 30-34 years: 0.98%; 35-39 years: 1.41%)

Hurley et USA Population-based 3118 Advanced paternal age not associated with a al., 2017 retrospective cohort significant increase in the rates of involving both preeclampsia, preterm birth, fetal growth naturally-conceived restriction, congenital anomaly, genetic and ART disorder, or neonatal intensive care unit conceived-children admission among couples subjected to ART (all adjusted RR estimates 95% confidence intervals crossing the null value of 1) OR odds ratio; CI confidence interval; RR: risk ratio; ART: Assisted Reproductive Technology

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