The male contribution to infertility has traditionally been overlooked, or at best oversimplified. In recent years efforts have been made to optimize diagnostic and therapeutic techniques to maximize fertility outcomes. A renewed focus on the male partner has resulted in an increased understanding of both genetic and epigenetic changes within the male germline. Furthermore, single-nucleotide polymorphisms, copy-number variants, DNA damage, sperm cryopreservation, obesity, and paternal age have recently been recognized as important factors that play a role in male fertility. Developing a deeper knowledge of these issues could potentially lead to improved success with assisted reproductive technology.
Key points
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A diagnosis of male factor infertility is associated with epigenetic changes, which may affect reproductive outcomes and could potentially impact the health of future generations.
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Genetic mutations likely play a role in male fertility, but individual polymorphisms only contribute to a small percentage of all male infertility cases.
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Cryopreservation affects semen analysis parameters and sperm DNA integrity, but the clinical superiority of fresh sperm over frozen sperm has not been firmly established.
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Obesity among men of reproductive age is becoming increasingly prevalent and seems to have a detrimental impact on fertility potential.
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The role of paternal age on sperm quality and fertility outcomes is controversial and difficult to assess due to confounders arising from the female partner.
Introduction
The male partner’s role in infertility has been the subject of increased investigation over the last several years. Although the female partner has historically been the primary focus of an infertility evaluation, it is now clear that early recognition and treatment of male factor infertility substantially improves a couple’s chances of success with fertility treatment. Approximately 20% of couple infertility can be attributed solely to the male, and a male factor is believed to contribute at least partially to difficulties with achieving pregnancy in as many as 50% of infertile couples.
Since the birth of the first child conceived through in vitro fertilization (IVF) in 1978, physicians and researchers have made significant advancements within the field of infertility. In modern society, the use of assisted reproductive technology (ART) is now commonplace. Between 1987 and 2015, it was reported that 1 million babies were born through the use of IVF or ART in the United States, and the percentage of births arising from ART has been rapidly increasing. In 2015, 1.7% of all infants born in the United States and 4.5% of births in the state of Massachusetts resulted from ART. As of 2019, the total number of births achieved through ART likely exceeds 8 million globally.
The general population’s overall acceptance of IVF as a treatment modality likely stems from improvements which have been observed in IVF outcomes. IVF protocols have undergone a tremendous evolution over the years, resulting in successful family building for infertile couples. Optimization of both laboratory techniques and clinical practice has led to dramatic improvements in live birth rates after IVF. Based on preliminary data from the 2017 National Summary Report from the Society for Assisted Reproductive Technology, in women less than 35 years old using autologous oocytes, 46.8% of all initiated IVF cycles in the United States resulted in live births. This is a significant progress considering the IVF pregnancy rate of 6% originally reported by Edwards and colleagues in 1980. From the male perspective, technological advancements, such as an intracytoplasmic sperm injection (ICSI), first introduced in 1992, have made it possible for couples with severe male factor infertility or failed fertilization in previous IVF cycles to achieve pregnancy.
A recent trend within the field has been to minimize multiple gestations while increasing delivery rates and improving obstetric outcomes for singleton pregnancies. Attempts to achieve these goals have primarily focused on interventions related to the female partner or the IVF laboratory. Single embryo transfer at the blastocyst stage, the use of preimplantation genetic testing, and the concept of achieving embryo and endometrial synchrony through freeze-all cycles have been described as potential techniques to improve patient outcomes and have been incorporated into many clinical practices. To further improve IVF outcomes going forward, a focus on the male contribution to ART is crucial. This article will specifically highlight several topics related to male reproductive biology and will discuss how the genetic, epigenetic, and clinical aspects of male factor infertility are intrinsically linked to current IVF practice and the future success of IVF.
The relationship between epigenetics, transgenerational epigenetic inheritance, and in vitro fertilization
The term epigenetics was coined in the 1940s to describe interactions between genes and the environment that could not be fully explained through classic genetics. Today, the concept of epigenetics primarily refers to 2 major types of modifications that occur in chromatin: DNA methylation and posttranslational histone modifications. Epigenetic modifications are responsible for controlling numerous processes within humans and serve an important regulatory role within the male reproductive system. It is thought that the epigenetic remodeling that occurs during late spermiogenesis, primarily the sequential replacement of histones by protamines, protects sperm DNA from oxidative stress arising from exposure to the female reproductive tract.
As understanding of the sperm epigenome has increased, there has been a growing body of evidence supporting a link between abnormal epigenetic sperm methylation patterns and male factor infertility ( Fig. 1 ). Through the use of arrays or targeted sequencing after bisulfate conversion, various loci have been evaluated for associations with male infertility phenotypes. The results of these efforts have consistently demonstrated altered sperm acetylation and methylation patterns among men with oligozoospermia and oligoasthenoteratozoospermia when compared with normozoospermic controls. , ,
The relationship between epigenetics and infertility has also become a topic of public interest. A 2017 systematic review and meta-analysis received a great deal of media attention after authors reported a 50% to 60% decline in sperm counts among men in North America, Europe, Australia, and New Zealand between 1973 and 2011. Although this downward trend in semen analysis parameters is likely multifactorial or affected by confounders, lifestyle factors and the epigenetic changes which arise from environmental exposures, such as phthalates and bisphenol A are believed to contribute to the reported reduction in male fertility over the past several decades.
Because of the intrinsic link between male epigenetic markers and infertility, researchers have begun to investigate the potential use of the sperm epigenome as a prognostic tool for infertile couples. Currently, validation studies are underway to assess the accuracy of algorithms, which have been developed with the goal of predicting fertility outcomes based on methylation array data from sperm. Predictive algorithms related to the sperm epigenome may have practical benefits because studies have demonstrated that epigenetic aberrations in men may adversely affect early embryonic development. , Therefore, it is important to consider the possibility that men with epigenetic damage may experience diminished success with the use of ART as well as a potentially increased incidence of recurrent implantation failure or early pregnancy loss.
Although epigenetic changes may lead to diminished fertility, it has also been suggested that the use of ART per se can induce epigenetic changes, which may have detrimental effects on pregnancy outcomes and the health of offspring. Potential mechanisms by which IVF may lead to epigenetic changes include gamete handling, embryonic exposure to culture media, cryopreservation, and procedures, such as ICSI or trophectoderm biopsy for preimplantation genetic testing. , Theoretically, epigenetic changes arising from ART may also manifest as health consequences in future generations. Researchers have analyzed CpG sites within gene promoters of the placenta and umbilical cord in children conceived spontaneously and those conceived through IVF. These studies have shown that children conceived via IVF or ICSI possess epigenetic alterations in genes involved in disorders, such as obesity, type II diabetes, hypertension, cardiovascular function, and delayed growth velocity. , Although evidence exists supporting the idea that epigenetic changes arise from ART techniques, it is also important to consider the possibility that intrinsic maternal or paternal factors related to subfertility may be the true underlying cause of epigenetic abnormalities found in offspring achieved through ART.
In summary, epigenetic dysregulation that results in male factor infertility or which potentially arises from gamete manipulation and ART may also impact the health of future generations. Environmental exposures that alter epigenetic programming within the paternal germline may also transmit epigenetically altered patterns and phenotypes to future generations, even in the absence of ongoing environmental exposures. , Going forward, a clearer understanding of epigenetics is necessary to determine whether a true causal relationship exists between ART and epigenetic change. If such a relationship does exist, then optimization of IVF protocols to minimize the inheritance of epigenetic abnormalities should be an area of focus.
Single-nucleotide polymorphisms and copy number variants associated with male infertility
Multiple genetic causes of male factor infertility have been proposed. However, publications evaluating genetic etiologies of infertility have produced conflicting results. Studies have explored the possible relationship between autosomal genes, single-nucleotide polymorphisms (SNPs), copy number variants (CNVs) and their potential impact on spermatogenesis and ART outcomes. Although accumulating data support the important role of SNPs and CNVs in spermatogenesis, the effect of these variations on IVF outcomes remains to be determined and relatively few studies have investigated this subject.
A 2009 Dutch study investigated the relationship between infertility and single nucleotide changes in the genes NXF2, USP26, and TAF7L because these genes are believed to be crucial for spermatogenesis. Five autosomal genes (SYCP3, MSH4, DNMT3L, STRA8, and ETV5) were also evaluated. It was determined that changes in STRA8 and ETV5 were detected in a population of infertile men but not in a control group of men with normozoospermia. However, no other changes seemed to be linked to male infertility. Although the significant findings involving STRA8 and ETV5 were initially promising, a subsequent functional analysis revealed that alterations in these genes (as well as in the other genes assessed) were unlikely to cause infertility in men.
A 2012 study evaluated the possible association of 9 SNPs located on 8 different genes (FASLG, JMJDIA, LOC203413, TEX15, BRDT, OR2W3, INSR, and TAS2R38) with male infertility. Using multiplex polymerase chain reaction/SNaPshot analyses followed by capillary electrophoresis, the study authors found that 3 of the 9 SNPs were significantly associated with male infertility (rs5911500 in LOC203413, rs3088232 in BRDT, and rs11204546 in OR2W3). However, a 2017 case-control study failed to demonstrate any reliable associations between the TP53 gene and male infertility. Similarly, an SNP of rs4880 of the SOD2 gene was found to have no association with male infertility in a study of 519 men with idiopathic infertility and 338 fertile controls. Taken as a whole, it seems that although some SNPs have shown potential associations with infertility, others have not, and each individual SNP is unlikely to contribute in a significant fashion to male factor infertility in the larger sense. One of the major challenges with establishing associations between SNPs and infertility is that thousands or even tens of thousands of cases and controls would be required to generate strong conclusions. The feasibility of conducting this type of large-scale research has limited the current understanding of this topic.
CNVs within specific genes have also been proposed as a cause of male infertility. A 2019 publication reported that CNVs in cation channel of sperm (CATSPER) genes are associated with idiopathic male infertility in the setting of normal semen parameters. The application of array comparative genomic hybridization has been used to demonstrate that an increased number of specific distributions of CNVs may result in defective recombination and meiotic dysregulation. CNVs may also result in altered gene transcription and protein functioning, ultimately contributing to spermatogenic failure.
It is highly likely that genetic mutations play a role in male fertility, but each individual polymorphism may only contribute to a small percentage of male infertility cases. Because of this, testing for SNPs in the general infertile population has not gained clinical applicability. In the future, it may be important to identify specific genetic alterations within the infertile male population because certain genetic etiologies of infertility may affect prognosis or outcomes with ART. Currently, there is insufficient data linking SNPs or CNVs to ART outcomes because the power to detect these associations requires extremely large numbers of patients. The development of datasets incorporating genome-wide information from multiple institutions will likely be necessary to answer the important questions regarding the relationship between SNPs, CNVs, and clinical outcomes with IVF.
DNA damage
Traditionally, the semen analysis has been the cornerstone of a male fertility evaluation. Despite its widespread use, routine semen analysis cannot measure the fertilizing potential of spermatozoa, and semen analysis parameters do not account for functional sperm characteristics. Therefore, there has been a high level of interest related to the development of accurate tests, which predict sperm function and a semen sample’s ability to achieve pregnancy. The level of sperm DNA damage has been studied with the goal of increasing the diagnostic sophistication and predictive value of tests before IVF and ICSI. Incomplete apoptosis, the posttesticular environment, reactive oxygen species (ROS), and prolonged periods of abstinence are all proposed mechanisms by which sperm DNA damage may occur. Reported associations between DNA damage and diminished reproductive outcomes has led to the use of sperm DNA integrity testing in many clinical practices.
There are a variety of assays that can measure sperm DNA damage, including the single-cell gel electrophoresis (Comet) assay, the sperm chromatin dispersion (SCD) assay, the sperm chromatin structure assay (SCSA), and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling (TUNEL) assay ( Table 1 ). Although each test possesses inherent advantages and disadvantages, TUNEL is arguably the most variable and has been difficult to standardize, although recently the TUNEL assay using a benchtop flow cytometer has been standardized and validated. , Both the SCD and SCSA methods are indirect assays, which only detect single-stranded DNA breaks and involve acid denaturation. The Comet assay is labor intensive, requires a fresh semen sample, and lacks a standardized protocol. Unfortunately, to date, a perfect test does not exist, and the correlation between sperm DNA fragmentation and clinical outcomes remains somewhat questionable.
| Assay | Type of Assay | Year Introduced | DNA Breaks Detected | Commercial Assay Available? | Specimen Type | Advantages | Disadvantages |
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| SCD | Indirect | 2003 | Single-stranded DNA (ssDNA) | Yes | Fresh or frozen |
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| SCSA | Indirect | 1980 | ssDNA | No | Fresh or frozen |
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| Comet gel electrophoresis | Direct | 1998 | ssDNA double-stranded DNA (dsDNA) | No | Fresh |
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| TUNEL | Direct | 1993 | ssDNA dsDNA | Yes | Fresh or frozen |
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Traditional medical thinking as it relates to sperm DNA damage supported the idea that the epididymal environment protected spermatozoa and promoted the maturation of sperm. However, animal studies from the early 2000s contradicted these beliefs by reporting higher levels of DNA damage and decreased fertilization rates in sperm harvested from the epididymis or ejaculate compared with surgically extracted sperm from the testicle itself. The role of the epididymis in sperm DNA fragmentation was further investigated by Gawecka and colleagues, who reported that fluid from within the epididymis and vas deferens activates sperm chromatin fragmentation in a murine model.
In humans, publications have demonstrated lower levels of DNA fragmentation in testicular sperm, and some authors have documented higher live birth rates with ICSI in patients who used testicular sperm as opposed to ejaculated sperm. , However, the exact etiology of sperm DNA damage remains unknown, and studies comparing reproductive outcomes with epididymal and testicular sperm are contradictory and inconclusive. Despite a body of evidence supporting the epididymis as the site where DNA damage accrues in spermatozoa, human studies have failed to demonstrate superiority of testicular sperm to produce higher fertilization rates or live birth rates. , A 2018 meta-analysis demonstrated lower clinical pregnancy rates and fewer high-quality embryos in patients with high degrees of DNA fragmentation, but no significant difference in live birth rates. This meta-analysis also highlighted one of the primary limitations with current research regarding associations between sperm DNA damage and pregnancy outcomes, specifically the heterogeneity of studies and the use of multiple sperm DNA testing platforms, which often lack standardization. , Similarly, a meta-analysis from 2016 demonstrated a lack of predictive value for the TUNEL assay, SCD test, and Comet assay and reported no relationship between test results and IVF/ICSI outcomes. Although damage to sperm DNA may certainly play a role in ART success and a couple’s fertility potential, testing for sperm DNA fragmentation has not yet resulted in meaningful improvements in clinical outcomes.
Fresh versus frozen sperm
Since the first published report of human sperm freezing in 1957, cryopreserved sperm has become an integral component of reproductive medicine and modern infertility practice. In addition, the cryopreservation of sperm has become a standard way to bank gametes in oncology patients and in patients undergoing vasectomy. Cryopreservation is also an essential aspect of sperm donation programs. From a logistical standpoint, cryopreservation of sperm has many advantages. However, the use of fresh versus frozen sperm for fertilization in ART is an area of significant debate.
There is very little consensus within the literature regarding the impact of cryopreservation on reproductive outcomes after conventional IVF or ICSI. When either fresh or cryopreserved sperm is used for fertilization, samples have most frequently been obtained from the ejaculate. Because of several patient factors, however, it is not uncommon for spermatozoa to be obtained from the testes. Studies have addressed the use of fresh compared with frozen sperm as well as ejaculated versus testicular sperm. Despite this relative abundance of research, results have been contradictory. ,
Several publications have reported that cryopreservation does not detrimentally affect outcomes. For example, in 1996, Gil-Salom and colleagues reported no difference in fertilization rate, cleavage rate, or embryo morphology when comparing cryopreserved and fresh testicular spermatozoa in a population of men undergoing ICSI. Similarly, Ben-Yosef and colleagues in 1999 reported similar outcomes with fresh and cryopreserved sperm in men with nonobstructive azoospermia (NOA) undergoing testicular sperm extraction (TESE) procedures. The authors suggested that performance of TESE followed by sperm cryopreservation before the initiation of ovarian stimulation should be considered first line treatment and would allow for more adequate patient counseling based on TESE findings without sacrificing pregnancy outcomes. Publications evaluating the use of cryopreservation with ejaculated sperm have also demonstrated that cryopreservation of spermatozoa from men with poor sperm quality does not negatively affect fertilization and pregnancy rates after ICSI.
Conversely, other studies have documented clear correlations between cryopreservation of sperm and diminished membrane integrity, viability, and motility. The mechanical and osmotic stress associated with cryopreservation have also been linked to abnormal morphology, and an increase in ROS related to the freezing process has been reported to induce DNA fragmentation. In a recent publication by Schachter-Safrai and colleagues, it was determined that in cases of cryptozoospermia, frozen-thawed ejaculated sperm is inferior to fresh ejaculated sperm based on a comparison of fertilization rates. However, in men with NOA, no major differences were found between fresh and frozen-thawed testicular sperm. A 2004 publication reported that in a population of men undergoing ICSI, cryopreservation of sperm resulted in higher fertilization rates but lower embryo quality, lower pregnancy rates, and lower delivery rates. Taken as a whole, the existing literature remains inconclusive.
During the cryopreservation process, cryoprotectant agents, such as glycerol, ethylene glycol, dimethyl sulfoxide and dimethylformamide are incorporated into freezing protocols to minimize damage to the spermatozoa during the freeze-thaw process. Despite the use of cryoprotectants, the formation of intracellular ice crystals, toxicity related to the cryoprotectants themselves, and factors of osmotic, mechanical, and oxidative stress all contribute to loss in sperm motility, decreased survival during the thawing process, and aberrant intracellular calcium concentrations.
Although vitrification is now the most frequently used method to store oocytes and embryos, this method has been difficult to use in spermatozoa due to the relatively high concentrations of permeable cryoprotectants required. Recently, publications reporting novel vitrification protocols have shown improved sperm survival rates, higher motility, and lower levels of DNA fragmentation when compared with conventional slow freezing of sperm. , Use of alternative cryoprotectant agents, such as sucrose have also been proposed as a potential way to improve sperm motility, viability, and mitochondrial membrane potential integrity when coupled with vitrification.
Despite continued controversy regarding potential differences in outcomes after the use of fresh or frozen sperm, optimization of vitrification techniques for sperm samples may prove to be important in clinical practice in the years to come. Vitrification may ultimately result in improved semen parameters for cryopreserved specimens. In cases of severe male factor infertility, azoospermia, or in situations where only small numbers of spermatozoa are available for cryopreservation, vitrification could provide a viable alternative to conventional slow freezing. At present, semen analysis parameters from fresh specimens are generally superior to parameters using frozen sperm, although any long-term clinical advantages of fresh specimens over frozen remain to be determined.
Obesity and sperm epigenetics
In the United States, the prevalence of obesity in men of reproductive age has tripled since the 1970s, currently affecting greater than 33% of the adult population. Increasing rates of obesity have coincided with reports of decreased sperm quality and rising rates of male factor infertility. , The relationship between obesity and male factor infertility is multifactorial, but epigenetic alterations in sperm are thought to be induced by obesity and lifestyle. These epigenetic abnormalities may negatively affect embryogenesis and the health of offspring.
In the context of obesity, epigenetic programming seems to be altered in men with raised body mass indices ( Fig. 2 ). A 2016 publication by Soubry and colleagues demonstrated that men who are overweight or obese exhibit traceable alterations within the sperm epigenome. Specifically, lower methylation percentages at the MEG3, NDN, SNRPN, and SGCE/PEG10 differentially methylated regions exist in obese men when compared with lean controls. The finding of alterations within imprinted genes and methylation abnormalities within male gametes provides a useful foundation for ongoing studies investigating the relationship between obesity and epigenetic changes.
