Genetics of Mitochondrial Dysfunction and Infertility

Leigh A M Demain1,2, Gerard S Conway,3,4 William G Newman

1. Manchester Centre for Genomic Medicine, St. Mary's Hospital, Manchester Academic Health Sciences Centre (MAHSC), Manchester UK.2. Division of Evolution and Genomic Sciences, School of Biological Sciences, University of Manchester3. Department of Endocrinology, University College London Hospitals, London, UK.4. Institute for Women's Health, University College London Hospitals, London, UK.

Correspondence toProf William NewmanManchester Centre for Genomic Medicine, St. Mary's Hospital, ManchesterM13 9WL

T. 44 161 276 6276

Email William.newman@manchester.ac.uk

Abstract

Increasingly, mitochondria are being recognised as having an important role in fertility. Indeed in assisted reproductive technologies mitochondrial function is a key indicator of sperm and oocyte quality. Here, we review the literature regarding mitochondrial genetics and infertility. In many multisystem disorders caused by mitochondrial dysfunction death occurs prior to sexual maturity or the clinical features are so severe that infertility may be under-reported. Interestingly, many of the genes linked to mitochondrial dysfunction and infertility have roles in the maintenance of mitochondrial DNA or in mitochondrial translation. Studies on populations with genetically uncharacterised infertility have highlighted an association with mitochondrial DNA deletions, whether this is causative or indicative of poor functioning mitochondria requires further examination. Studies on the impact of mitochondrial DNA variants present conflicting data but highlight POLG as a particularly interesting candidate gene for both male and female infertility.

Introduction

Mitochondria are known as the powerhouse of the cell and are present in varying numbers in every nucleated eukaryotic cell. Apart from the most well-known mitochondrial role of oxidative phosphorylation, generating ATP, mitochondria also have roles in additional pathways including apoptosis, FeS cluster generation, ion homeostasis and steroid hormone biogenesis (1). Mitochondria are unique organelles as they are comprised of proteins encoded by both the mitochondrial and the nuclear genomes. The small mitochondrial genome codes for 13 polypeptides, with the majority of proteins required for mitochondrial function encoded by the nuclear genome. Mitochondrial DNA is inherited exclusively maternally and is present in multiple copies in a cell (1). Multiple copies of mitochondrial DNA per cell can result in mitochondrial DNA heteroplasmy, where a variant in mitochondrial DNA may be present in varying allele frequencies within a cell (1). With pathogenic variants this often means that the load of the variant must pass a certain threshold in a specific tissue before a phenotype is seen (2). Many genetic conditions have been linked to mitochondrial dysfunction with an increasingly wide phenotypic spectrum, from very severe phenotypes causing childhood death to milder adult onset conditions restricted to a single organ, such as Leber’s hereditary optic neuropathy (2). Some of these phenotypes include infertility.

Infertility is a common morbidity, affecting up to 7% of all couples. Almost 50% of infertility is estimated to be attributable to genetic factors (3). Genetic factors causing infertility can include; chromosomal abnormalities, single gene disorders and polygenic disorders (3). One prominent cause of female infertility is primary ovarian insufficiency (POI), also known as premature ovarian failure, defined as the cessation of the menses before 40 years of age, with raised gonadotrophins, and affects approximately 1% of women. This compares to the

average age of menopause in western populations of 51 years. The age of presentation of POI is highly variable, with individuals presenting with primary amenorrhea and absent or streak gonads to those with secondary amenorrhea and infertility. Some women with POI may bear children before the onset of secondary amenorrhea (4). Male infertility accounts for more than 50% of the reported cases of infertility. The diagnosis of male infertility may be based on the reduced number or poor function of sperm. Idiopathic male infertility where the sperm falls under normal parameters is also reported (5). Many cases of POI and male infertility have an unknown aetiology (3).

Identifying the genetic basis of infertility is important in order to develop treatments and potentially improve the outcomes of assisted reproductive technology. The functional role of mitochondria is becoming an increasingly important consideration in both male and female fertility. In assisted reproductive technologies mitochondrial function is a key indicator of sperm and oocyte quality (6, 7). Mitochondrial function has also been proposed as an important factor in ovarian aging (8). While mitochondrial DNA is inherited maternally, variants in many nuclear encoded genes can cause mitochondrial disorders and so all forms of inheritance should be considered in examining relationships between infertility and mitochondrial dysfunction (2). Here, we present an overview of the genetics of mitochondrial dysfunction related to infertility.

Mitochondria in the Reproductive System

As with many tissues the reproductive organs have specific metabolic and energy requirements during development and during adult function. The mature oocyte is the largest cell in the human body and has a correspondingly high mitochondrial content. Numbers of mitochondria and their function are tightly linked to the development of the oocyte from primordial germ cells (9). Mitochondria are incapable of de novo biogenesis and must be generated by expansion and fission from the existing mitochondria in a cell. The mitochondria from sperm are actively excluded from the zygote and as such all mitochondria and therefore all mitochondrial DNA is inherited from the cytoplasm of the fertilised mature oocyte (1).

Oocytes are formed initially from primordial germ cells. These cells have a low mitochondrial content of approximately 10 per cell due to a cessation of mitochondrial biogenesis during very early development causing the existing mitochondria in the fertilised oocyte to be split between the daughter cells causing a bottleneck with each daughter cell having a distinct set of mitochondrial DNA with differing levels of heteroplasmy for variants. The bottleneck with the massive reduction of mitochondrial content of the oocyte is important to generate a homogenous population of mitochondrial DNA in essentially a form of asexual reproduction (8, 10). Hypomorphic variants are removed via this mechanism while more deleterious variants seem to circumvent it in a process that is not yet understood (8).

As the oocytes develop the mitochondrial content increases alongside the cytoplasmic volume so that the number of mitochondria in the mature oocyte is approximately 5 million, the highest of any cell. The number of oocytes increases to peak at approximately 7 million at week 20 of the developing embryo. After this the number of oocytes is continuously and drastically depleted via atresia to approximately 300,000 at menarche (11). The depletion of oocytes and follicles continues throughout a woman’s life, most follicles that start to mature during each monthly ovarian cycle are also lost due to atresia and therefore the vast majority of the original oocytes in the ovary are destined to be lost. Mitochondria may play an important role in the depletion of oocytes by apoptosis (8). Data from the BAX null mouse indicates that the mitochondrial apoptotic pathway is involved in the atresia of the oocytes (12).

Spermatogenesis unlike oogenesis is a continuous process throughout life resulting in a theoretically unlimited number of gametes. Even though spermatozoal mitochondria are not transferred to the zygote, mitochondria have an important role in the development and function of the male reproductive system. The developing testes have very high energy requirements and, as with the ovaries, the mitochondrial function and morphology are tightly linked to the sperm developmental stage (13). The testes have specific isoforms of mitochondrial proteins highlighting their distinct metabolic requirements. During the final maturation stages of sperm much of the cytoplasm and mitochondria are lost and the remaining mitochondria are concentrated around the sperm mid-piece where they are likely to be vital for sperm motility and therefore male fertility (14, 15).

Mitochondria have functions that are not directly associated with the respiratory chain complexes which may affect fertility. The most noticeable of these is the role in steroid biogenesis. The oocyte mitochondria also are responsible for regulating calcium waves that are essential in the developing zygote (1).

Predicted outcomes of mitochondrial dysfunction in the reproductive system

As mitochondria have a large role in the development and function of the reproductive system we could expect mitochondrial dysfunction to disrupt the normal function of the ovaries and or sperm. An important investigative tool for this is the use of animal models, especially mice. Some mouse models, whether developed as a model of human disease or identified from random knockout and the subsequent phenotypic screens, are detailed below.

ANT4 encodes an adenine nucleotide transferase involved in ADP/ATP exchange across the mitochondrial inner membrane. ANT4 is selectively expressed in both human and murine testicular germ cells and is necessary for the translocation of ATP from the mitochondrial matrix into the cytosol. Disruption of murine Ant4 leads to the complete loss of male fertility (16). The male germ cells are subject to meiotic arrest with no spermatids or mature spermatozoa, both of which are haploid stages, present in the testes. This suggests that ATP

and consequently functioning mitochondria are required for the maturation of murine sperm (17). Conversely, Ant4 is expressed in the developing murine ovary but is not required for oogenesis. Female mice with disrupted Ant4 were fertile with only slightly smaller litter sizes than their wild type counterparts (18).

Mice of both sexes with disruption of Immp2l have fertility defects. IMMPL2 encodes Immpl2 (inner mitochondrial membrane peptidase 2 like), part of the mitochondrial inner membrane peptidase complex which cleaves sorting signals. The mice had a mutation which resulted in undetectable levels of Immpl2 transcripts. Homozygous female mice were infertile and had defects in folliculogenesis and ovulation. Male mice were sub-fertile, had erectile dysfunction and age-related defects in spermatogenesis (19). In male mice this defect in spermatogenesis, defined as a reduction in germ cells, was associated with an increase in oxidative stress and increases apoptosis in the germ cells at all stages. The mice showed other age associated defects such as ataxia but no increase in the number of mitochondrial DNA mutations in sperm. It was proposed that an increase in ROS, and as such oxidative stress, resulted in the age related defects in spermatogenesis in the homozygous variant male mice (20).

The knockout mouse for Clpp is characterised by hearing loss, neurological problems and infertility in both sexes (21). CLPP encodes a subunit of CLPXP, a mitochondrial protease. The mouse model mirrors the human phenotype of Perrault syndrome (MIM 614129, sensorineural hearing loss and POI in 46 XX karyotype females) caused by variants in CLPP and other nuclear encoded mitochondrial genes (22). The male infertility in the mouse model was considered to be a difference from the human phenotype, but recently azoospermia was identified in a male with Perrault syndrome due to variants in CLPP (21, 23).

Nakada et al. (24) created a mouse model with a large mitochondrial DNA deletion, of 4696 base pairs, present at varying levels of heteroplasmy. The mice were split into three groups depending on phenotype and the levels of the mutant mitochondrial DNA. Group one with <68% mutant DNA showed a wild type phenotype. Group two with 70-80% mutant mitochondrial DNA had a minor mitochondrial respiratory deficiency after glucose loading. Group three with >80% mutant mitochondrial DNA was affected by a multisystem mitochondrial disease phenotype with symptoms including lactic acidosis, myopathy, renal failure and deafness. During male fertility assessment, group two showed reduced progeny in comparison to group one and the wildtype mice despite having no overt mitochondrial phenotype. Group three demonstrated no successful matings due to behavioural defects. Assessment of the sperm of group two and three showed morphological defects and a reduction in both sperm motility and number. In vitro fertilisation assays showed a reduction in fertilisation rates in group two and no successful fertilisations in group three. Further testing revealed that the spermatocytes were arrested ant the meiotic stage and removed via apoptosis. Interestingly they showed that in these mice a moderate level of

mutant mitochondrial DNA is enough to reduce fertility and cause sperm dysfunction without causing an overt mitochondrial phenotype. Female mice were shown to be fertile even when carrying very high levels of mutant mitochondrial DNA and showing a severe mitochondrial phenotype (24).

Mice homozygous for a knock in mutation, ablating the proof reading function of the mitochondrial DNA polymerase subunit, in PolgA had a premature aging phenotype, including reduced fertility. The mice showed significantly increased point mutations in mitochondrial DNA as well as increased mitochondrial DNA deletions. Fertility in both sexes was greatly reduced with females being infertile after 20 weeks of age and a total absence of sperm in male mice greater than 40 weeks of age (25). Variants in POLG in humans have been associated with syndromes characterised by mitochondrial DNA depletion and deletions, some of the affected individuals have fertility problems (26).

Mitochondrial disorders associated with infertility

Mitochondrial disorders are a consequence of mitochondrial dysfunction and can be caused by both defects of mitochondrial DNA and of nuclear genes (2). Mitochondrial disorders have a wide phenotypic spectrum and may affect individual organs, such as in mitochondrial DNA linked non-syndromic hearing loss (27), or may present with severe multisystemic disease. Many mitochondrial disorders have overlapping phenotypes and may be difficult to distinguish clinically (2). A number of mitochondrial disorders have been linked to infertility.

Variants in Mitochondrial DNA

Variants in mitochondrial DNA can result in multiple different phenotypes (2). However, in few have infertility been reported.

Kearns Sayre syndrome (KSS, MIM 530000) is caused by large-scale deletions of mitochondrial DNA. The most common deletion associated with KSS is a 4977 bp deletion. KSS is characterised by progressive external ophthalmoplegia (PEO), pigmentary retinopathy and at least one of the following: cardiac conduction block, cerebellar ataxia or high concentrations of protein in the cerebrospinal fluid by 20 years of age (28). Additional clinical features of KSS include hypogonadism and irregular menses. Hypogonadism is present in approximately 20% of subjects with KSS with clinical features supporting the diagnosis (29) but the prevalence in genetically confirmed cases has not been reported. Therefore, the frequency of hypogonadism in genetically uncharacterised subjects with KSS may be overestimated as some of the subjects could have a mitochondrial DNA deletion syndrome, which results in a similar phenotype to KSS including infertility (28).

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS, MIM 540000) is caused by variants in mitochondrial DNA the most common of which is the m.3243A>G variant in the gene encoding mitochondrial tRNALeu(UUR) (MIM 590050). Clinical features include stroke-like episodes, epilepsy, lactic acidemia, myopathy, recurrent

headaches, dementia and hearing impairment. Endocrine dysfunction, including diabetes and growth hormone deficiency, is also common in individuals affected by MELAS (30). A number of studies have reported a link between MELAS and low levels of gonadotropins and estradiol, most of the cases in these studies have not been genetically confirmed (31, 32). In several studies women with the m.3243A>G variant had low levels of gonadotrophins. In most cases, as in many of the genetically unconfirmed cases of MELAS the hypogonadism was attributed to defects of the hypothalamic pituitary axis (32, 35, 36). In a male subject with the m.3243A>G variant in mitochondrial DNA and recurrent strokes high levels of the variant in sperm correlated with low sperm motility (37). A subject with an m.12147G>A change in mitochondrial tRNAHis (MIM 590040) displayed a phenotype of hypogonadotropic hypogonadism, stroke-like episodes, ataxia, dementia and myoclonic epilepsy (38) highlighting that hypogonadism in MELAS is not exclusively associated with the m.3243A>G variant.

A variant, m.4296G>A, in mitochondrial tRNAIle (MIM 590045) was proposed to be pathogenic in a male subject with Leigh like syndrome, Parkinsonism and hypogonadism with developmental delay. The proportion of the variant in the subject was 95% whereas the proportion in the subject’s unaffected mother was 58% (39).

In a single family a variant in the overlapping section of MT-ATP6 (MIM 516060) and MT-ATP8 (MIM 516070) was associated with a phenotype which included hypergonadotropic hypogonadism (40). The affected female proband and her male sibling displayed a phenotype of hypergonadotropic hypogonadism, ataxia, peripheral neuropathy and diabetes mellitus. The variant m.8561C>G was present with >99% heteroplasmy in the blood of the affected siblings (40).

Mitochondrial DNA depletion syndromes

Mitochondrial DNA depletion syndromes are caused by variants in nuclear genes and result in large scale mitochondrial DNA deletions or depletion (2).

POLG (MIM 174763) encodes the catalytic subunit of DNA polymerase gamma, the mitochondrial DNA polymerase (26). Variants in POLG can cause a spectrum of disorders, including both autosomal dominant and recessive PEO, characterised by deletions or depletion of mitochondrial DNA (26). Recessive and dominant variants in POLG have been associated with a phenotype of PEO, parkinsonism and premature menopause. Many of the affected women described with this condition have hypergonadotropic hypogonadism (26, 41). In three families with autosomal dominant inheritance the phenotype was associated with a heterozygous p.Y955C variant (41). Testicular atrophy was also reported in one of the families with a heterozygous Y955C variant (26). Further, there has also been a woman with heterozygous POLG variant with muscle atrophy, cataracts, ovarian dysgenesis and 3-methylglutaconic aciduria (42). However, infertility is not commonly associated with other POLG syndromes.

Biallelic variants in RRM2B (MIM 604712), encoding ribonucleotide reductase M2B which catalyses synthesis of dNTPs, causes a severe multisystemic phenotype, including PEO, ptosis, muscle weakness and sensorineural hearing loss (43). Hypogonadism has been reported in three individuals with recessive variants in RRM2B (43, 44).

C10orf2 (MIM 606075) encodes twinkle, a mitochondrial DNA helicase (45). Biallelic variants in C10orf2 are associated with infantile onset spinocerebellar ataxia (IOSCA, MIM 271245), characterised by ophthalmoplegia, hearing loss, ataxia, epilepsy, sensory neuropathy, cerebellar atrophy and hypergonadotropic hypogonadism in females (46). Subjects with IOSCA have mitochondrial DNA depletion in liver and brain tissue (47). Recessive variants in C10orf2 have also been identified in women with Perrault syndrome (MIM 233400) (48). However, these individuals as well as having sensorineural hearing loss and POI characteristic of Perrault syndrome also had significant neurological impairment consistent with mitochondrial DNA depletion. Heterozygous variants in C10orf2 have been reported in individuals with PEO, depression, neuropathy, ataxia and hearing loss (49). While hypergonadotropic hypogonadism is a clinical feature of IOSCA (47) it is only rarely a feature of PEO due to C10orf2 variants. One case of ovarian failure was reported in a study of 33 individuals with PEO due to variants in C10orf2 (49). Fertility appears unaffected in males with variants in C10orf2.

Biallelic variants in MPV17 (MIM 137960) are associated with lethal infantile neurohepatopathy, a mitochondrial DNA depletion syndrome (MIM 256810) (50). The mpv17 protein is located on the mitochondrial inner membrane but its function is currently unknown (50). Recently adult onset cases have been reported in association with recessive variants in MPV17. In one a 25 year old woman had secondary amenorrhea, and was homozygous the missense variant p.P98L in mpv17 (51). However there is insufficient evidence that infertility in women is a part of the phenotypic spectrum.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE, MIM 603041) is an autosomal recessive condition characterised by ptosis, PEO, gastrointestinal dysmotility, cachexia, peripheral neuropathy, and leukoencephalopathy. Subjects typically have mitochondrial DNA depletion and deletions detectable in muscle tissue (52). MNGIE is caused by variants in TYMP (MIM 131222) encoding thymidine phosphorylase, which cause a nucleotide imbalance with elevated levels of thymine precursors and causes mitochondrial DNA instability (53). Hypergonadotropic hypogonadism has been described in two male subjects with MNGIE and confirmed TYMP variants (52, 54).

Variants in nuclear genes encoding mitochondrial proteins

Perrault syndrome (MIM 233400) is an autosomal recessive condition characterised by sensorineural hearing loss in both sexes and primary ovarian insufficiency (POI) in 46,XX karyotype females (55). Additional features, most commonly neurological, may be present in some affected individuals (56). Perrault syndrome is genetically heterogeneous (57) with

biallelic variants reported in five genes to date; HSD17B4 (MIM 601860) (58), HARS2 (MIM 600783) (59), LARS2 (MIM 604544)(60), CLPP (MIM 601119)(61) and C10orf2 (48). Of the five genes only HSD17B4, a peroxisomal enzyme (62), does not have a known mitochondrial function. HARS2 and LARS2 encode mitochondrial histidyl and leucyl tRNA synthetase respectively, the proteins responsible for attaching the cognate tRNA to the appropriate amino acid (63). CLPP encodes caseinolytic protein peptidase a subunit of the mitochondrial protease CLPXP complex (64). C10orf2 encodes twinkle, a mitochondrial DNA helicase (45). POI in Perrault syndrome presents as primary or secondary amenorrhea and a hormone profile consistent with hypergonadotropic hypogonadism. However there is ascertainment bias as most women reported to date have absent, streak or small ovaries (22). The effect of Perrault syndrome on male fertility is unclear and in most cases it is presumed to be normal. Males with Perrault syndrome due to HARS2 variants, from a single family, have had children (59). Recently a case was reported of a male with Perrault syndrome, due to variants in CLPP, with azoospermia (23). The fact that azoospermia is reported in the clpp null mouse suggests that azoospermia is an un- or under-recognised feature of Perrault syndrome. Males are under-ascertained for this condition due to the sex-limited phenotype and full investigations into male fertility are rarely performed (23).

AARS2 (MIM 612035) encodes mitochondrial alanyl tRNA synthetase responsible for attaching alanine to the cognate tRNA in the mitochondrial matrix (65). Recessive variants in AARS2 were associated with leukoencephalopathy and POI in six subjects (MIM 615889) (66). Subjects had childhood onset of neurological deterioration including ataxia, spasticity, cognitive decline and white matter abnormalities. The five female subjects all presented with either primary or secondary amenorrhea due to POI. The fertility of the single affected male was not reported. As variants in HARS2 and LARS2 cause POI it may be that variants in other mitochondrial aminoacyl-synthetase genes result in POI.

Non-syndromic variants associated with infertility

Mitochondrial proteins encoded by both mitochondrial and nuclear DNA are good candidates to cause infertility. Variants in novel genes or hypomorphic alleles of some of the genes detailed above could be causative.

Despite the association of POLG and infertility in mitochondrial disorders and the murine phenotype in a non-syndromic cohort only one heterozygous variant was found in 201 women with POI (67). Duncan et al did not identify p.Y955C or any of the three most common pathogenic variants in POLG in a cohort of 57 women with POI (68). The results of these two studies are not too surprising, as women with these genotypes would be expected to have a more severe phenotype. However, a broader screen of POLG did not identify any putative pathogenic variants in POI (69). However polymorphisms in POLG have been associated with both the age of menopause and early menopause in large meta-analyses of genome wide association studies (GWAS) (70, 71).

POLG contains a CAG repeat region at the 5’ end for which 10 repeats is considered the common allele (14, 72). A number of studies have been performed on the association between the POLG CAG repeat region and male infertility but the evidence is conflicting. An association was found between infertility in men and homozygosity for CAG repeats, i.e. not 10 repeats (72). This association was supported by a study in Danish men showing the relationship between the lack of the common allele and idiopathic infertility (73). A higher incidence of heterozygosity for CAG repeats associated with increased mitochondrial DNA load was found in men with very low quality sperm. The authors were unable to link this to a defect of mitochondrial DNA maintenance and note that the significance remains unclear (14). Conversely, two studies found no association with the lack of the common allele in French and Italian infertile men (74, 75) and another study found increased incidence of heterozygosity for variant alleles in fertile men (76).

Small studies have reported links between mitochondrial DNA variants and POI. One study reported increased numbers of mitochondrial DNA variants in women with POI (77). In addition the POI population had higher levels of reactive oxygen species (ROS) than the controls (77). Significantly higher levels of ROS in POI subjects were also reported, as well as an increase in missense variants in MT-ATP6 (78). A higher number of missense variants in MT-CO1 (MIM 516030) were also reported in a small study of POI subjects (79). A similar study performed on infertile Tunisian men identified a novel variant, m.9387G>A, in MT-CO3 (MIM 516050) in three asthenozoospermic men not identified in the population matched controls (80). These studies have not been replicated in larger populations.

A number of studies have linked mitochondrial DNA deletions, including the common 4977 bp deletion, to a reduction in sperm motility and quality (81-84). The presence of the 4977 bp deletion negatively correlated with sperm motility (84), but the type of deletion was not predictive of aetiology (83). Whether the deletions are the primary cause of infertility in these subjects or are secondary to other mitochondrial dysfunction is unknown. It is clear though from these studies that damage to mitochondrial DNA correlates with reduced sperm motility and infertility.

Two studies have linked mitochondrial DNA depletion with female infertility. In women with impaired ovarian reserve (69) and with POI (85) mitochondrial copy number was significantly lower than in controls (69, 85). The causes of the depletion remain unclear.

Conclusion

Mitochondrial function has an important role in fertility. However, genetic studies regarding infertility associated with mitochondrial dysfunction are sparse. Many of the reported cases of infertility associated with mitochondrial syndromes have not been genetically confirmed. Given the range of phenotypic overlap between mitochondrial syndromes it is difficult to derive clear genotype-phenotype associations. In the most severe cases of mitochondrial syndromes with other dominant clinical features precluding a full fertility assessment and so

under reporting of the fertility phenotype is likely (86). This applies even more so for males who may not display the overt clinical signs of fertility as women in whom amenorrhea is a more apparent phenotype. Interestingly, many mitochondrial disorders associated with infertility are caused by variants that affect either mitochondrial DNA or the mitochondrial translation pathway. However, not all variants in this protein synthesis pathway cause infertility.

GWAS and other population studies have failed to identify any definitive relationships with variants in genes encoding mitochondrial proteins, but despite conflicting results POLG remains a good candidate gene for infertility in both men and women.

The pathogenesis of infertility in mitochondrial syndrome may be direct though germ cell damage or indirect through diminished gonadotrophin drive. In the case of gonadotrophin deficiency, fertility may be retrieved using ovulation induction with gonadotrophins therapy in both men and women. In the case of late onset ovarian insufficiency, it is important to identify those at risk at an early stage, as oocytes cryopreservation may be possible if sufficient ovarian reserve allows. For those with established ovarian failure or azoospermia, gamete donation will be required.

Infertility is genetically heterogeneous and mitochondrial dysfunction is not the only mechanism by which this complex phenotype arises. The variants linked with mitochondrial dysfunction and infertility may be rare, population specific or in non-coding regions. As technologies such as exome and whole genome sequencing become more widely used in studies of infertility it is likely that more mitochondrial variants associated with infertility will be identified and the pathogenesis more fully defined.

Table 1

Gene Study outline Cohort Main Points Reference

POLG

Screened women with POI for 5 known pathogenic variants in POLG 201 women with POI One heterozygous variant (p.R953C) was found in

one woman with POI (67)

Screened women with POI for p.Y955C and the three most

common pathogenic variants in POLG

57 British women with POI and no fragile X pre-mutations No variants identified. (68)

GWAS meta-analysis – age of menopause 38,968 women of European descent A SNP in POLG is associated with age at

menopause (71)

GWAS meta-analysis – early menopause

3493 women with early menopause13598 women with normal age of

menopause

A SNP in POLG contributes the risk of early menopause (70)

POLG and mitochond

rial DNA levels

Screened for variants in POLG and mitochondrial DNA levels

101 women with impaired ovarian reserve including 59 with POI

Control group - 43 women with intact ovarian reserve and 53 post-

menopausal women

No variants in POLG identified.Significant mitochondrial DNA depletion in the

women with impaired ovarian reserve.(69)

POLG CAG repeat region

Analysis of CAG repeats. The common allele is 10 repeats

99 infertile men98 fertile men

An association between the absence of the common allele and sperm defects (72)

Analysis of CAG repeats in Danish men with idiopathic infertility or

subfertility

429 infertile/ sub-fertile men869 fertile men

The absence of the common allele is more prevalent among the infertile/sub-fertile men

(73)

Analysis of CAG repeats and mitochondrial DNA content of

sperm

56 oligoasthenoteratozoospermic men

134 men with one or two sperm defects

80 normal men

A higher incidence of heterozygosity for CAG repeats associated with increased mitochondrial DNA load in oligoasthenoteratozoospermic men

(14)

Analysis of CAG repeats in Italian men

195 infertile men190 fertile men

No association with the lack of the common allele and infertility (74)

Analysis of CAG repeats in French men with idiopathic infertility

433 infertile men91 fertile men

No association with the lack of the common allele and infertility (75)

Analysis of CAG repeats in poor and normal sperm samples

182 poor sperm samples93 normal sperm samples

Increased incidence of heterozygosity for variant alleles in fertile men (76)

Mitochondrial DNA, 4977 bp deletion

Investigated the association of the deletion and reduced sperm

motility

19 infertile men21 fertile control men

Negative correlation between the presence of the mitochondrial DNA deletion of 4977 bp and sperm

motility(81)

Investigated the presence of the deletion in a blinded cohort

53 fertile men11 infertile men

The deletion does not correlate with the phenotype (87)

Investigated the association of the deletion with male infertility using

an improved assay

83 abnormal sperm samples31 normal sperm samples

Negative correlation between the presence of the mitochondrial DNA deletion of 4977 bp and sperm

motility(84)

Investigated the association of the deletion and reduced sperm

motility in the Iranian population

256 infertile men200 fertile men

There is a strong link with the presence of the deletion and asthenozoospermia in this

population(82)

Mitochondrial DNA deletions

Investigated the association of deletions and reduced sperm

motility

27 sub-fertile men9 fertile men

Negative correlation between the presence of mitochondrial DNA deletion, 4977 bp, and sperm

motility. Larger deletions found in very poor

(88)

quality sperm

Analysed 3 mitochondrial DNA deletions in sperm

9 fertile men39 infertile men with various sperm

defects

Multiple mitochondrial DNA deletions are associated with poor sperm quality but the type of

deletion isn’t predictive of the aetiology of the defect

(83)

Mitochondrial DNA

Six mitochondrial genes were examined for variants in infertile

Tunisian men and controls

34 asthenozoospermic men32 men with idiopathic infertility

100 fertile men

A novel variant (m.9387G>A, MT-CO3) was identified in three asthenozoospermic men but not in controls or men with idiopathic infertility

(80)

Quantified mitochondrial DNA in oocytes

17 women with POI16 women with ovarian dystrophy

14 control women

Mitochondrial copy number is significantly lower in the oocytes of women with POI (85)

MT-ATP6 Analysed the MT-ATP6 gene and ROS levels in women with POI

20 women with POI20 control women

ROS levels were significantly higher in women with POI

2 variants MT-ATP6 were significantly more common in the women with POI than in controls

(78)

MT-CO1 All mitochondrial DNA was analysed in women with POI

63 women with POI63 control women

The women with POI had a significantly higher number of missense mutations in MT-CO1 (79)

Table 1. Studies of the effects of mitochondrial variants on non-syndromic infertility.

ROS, reactive oxygen species. POI, primary ovarian insufficiency. Bp, base pairs. GWAS, genome wide association study.

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