4. Infertility: A Precedent of Male Reproductive Cancers?
It has been well documented that cancer as a pathological process may exhibit a variety of deleterious effects on male fertility, even before any treatment has been administered [119
]. The most common malignancy-associated effect reported is the disruption of the hypothalamic-pituitary-gonadal axis, leading to serious alterations to a delicate endocrine balance within the male reproductive system [121
]. Systemic effects of a tumor presence include a direct immunological or cytotoxic injury to the germinal epithelium [119
], leading to significant alterations of spermatogenesis [124
] and generation of antisperm antibodies [125
]. Fever and malnutrition commonly observed in cancer patients may be associated with spermatogenic alterations, leading to a severely diminished sperm concentration or even azoospermia [126
]. Psychological effects including anxiety and depression have been associated with infertility in many oncological cases. Sexual dysfunction and fertility distress are known to be long-term consequences proceeding for years after the actual cancer diagnosis [128
]. All the above-indicated pathological changes may individually or collectively lead to decreased semen quality and fertility impairment, being often present at the very time of diagnosis [119
Given shared etiologies, risk factors and molecular pathways, recent attention has been placed on the question if male reproductive dysfunction may precede testicular or prostate cancer. The remaining section of this review will therefore discuss the currently available research data interconnecting these diseases, limitations of the studies, as well as possible mechanisms emphasizing on their mutual relationship.
The development of TGCCs due to infertility has been studied in the past. It is believed that TGCCs could arise in men with underlying carcinoma in situ
(CIS) cells. Pryor et al.
] studied the relationship between infertility and TC development. Out of a pool of 2043 men, the study identified 8 patients presenting with CIS cells. 6 out of the 8 subjects eventually developed TGCCs later in life. Different reports suggest that the presence of CIS cells may lead to both infertility through impaired spermatogenesis as well as cancer as these cells have a genetic stem cell profile and pluripotent characteristics carrying the potential to proliferate into TGCCs [15
], supporting the idea that TC and infertility share similar environmental, genetic, and ethnic backgrounds [131
]. Petersen et al.
] provided additional evidence that patients diagnosed with TGCC and CIS cells usually present with abnormal semen parameters. A Danish study by Jacobsen et al.
] looked into a pool of patients seeking infertility treatment at a sperm analysis laboratory in Copenhagen. The investigation found that out of the 32,442 cases, 89 progressed to develop TC, meaning that men with abnormal semen parameters or infertility were at a 1.6 times higher risk to develop TC in comparison with healthy men. Out of the 89 cancer cases, 50 developed seminomas, 37 had nonseminomas and 2 were diagnosed with an unspecified testicular malignancy. Additionally, the study reports that men were more likely to be diagnosed with cancer within 2 years after the first semen analysis, suggesting that abnormal semen parameters were indicative of future risk of TC development. In the case of azoospermic patients, those who had no children faced a 3.65 higher chance of eventual TC development when compared to azoospermic patients with children. Furthermore, men with spermatozoa concentrations ranging from 0 to 20 million were more susceptible to TC occurrence compared to men with concentrations greater than 20 million (a standardized incidence ratio 2.3 vs.
1.1, respectively). It is however important to keep in mind that both patient groups ultimately faced the threat of possible TC development. At the same time, men with abnormal sperm motility were 2.5 times more susceptible to TC, while men with abnormal morphology had a 3 times higher risk of testicular tumor development. A combination of different semen abnormalities led to a drastic increase in a possible TC diagnosis, as two joint subfertility parameters resulted in a 2.7 times higher risk, while a fusion of all three abnormalities was associated with a 9.3 higher chance of future testicular tumor development. It is therefore important to acknowledge that patients diagnosed with 3 subfertility measures faced a 9-fold increase in eventual TC diagnosis. These results prove that suboptimal semen parameters could be a precursor as well as a predictor for TC, thus they should be taken seriously. As discussed in the study, Jacobsen et al.
] assume that a hypothetical removal of patients with infertility caused by a female factor would lead to even more significant differences. Doria-Rose et al.
] brought ingenuity to their methodology as instead of observing infertile men and conducting regular follow-ups, they looked directly at TC cases and subsequently used a “backtrack” strategy using the National Cancer institute’s Surveillance Epidemiology and End Result (SEER) in order to see whether or not the patients had been diagnosed with infertility prior to TC development. Out of 329 men, 183 were diagnosed with seminomas and 146 had nonseminomas—a finding consistent with the outcomes by Jacobsen et al.
]. 329 TC cases were more likely to have been previously diagnosed with infertility or had cryptorchidism. The study showed that the testicular malignancy was 2.4 times more likely to have been interrelated with previous infertility. The most important data collected from this study included a reduced risk of TC development in men who had previously fathered children. The odds ratio was 0.76 however the limitation of this observation was based on a comparative analysis with infertile men and patients diagnosed with cryptorchidism. Excluding cryptorchidic cases, the risk ratio was adjusted to 0.82 and to 0.87 when unmarried men were accounted for, which is nevertheless a significant conclusion clearly expressing that men with children or fertile men had lower chances of future TC development. Furthermore, the study showed that increasing number of children had no impact on a potential further decrease in risks. Walsh et al.
] conducted a patient study in California, US, using data collected from 22,562 patients. This pool included infertility cases due to male factors, female factors, as well as combined factors. The analysis identified men with male factor infertility using semen analysis. Using SEER the study compared specific findings of interest with data from an average population, matching categories of age and geographical location in order to close the margin for error. The authors found that infertile patients with male factor infertility were at a 2.8 higher risk for eventual TC development compared to the average population, validating conclusions from previous studies. Data from studies examining the relationship between testicular cancer and male infertility are summarized in Table 1
Epidemiologic studies focused on the association between male infertility and testicular cancer.
Epidemiologic studies focused on the association between male infertility and testicular cancer.
|Author(s)||Country and Year||Design||Subjects||Finding(s)||Conclusions|
|Pryor et al. ||UK 1983||Case study||2043 males from infertile couples who underwent testicular biopsy from 1955 to 1982.|
Carcinoma in situ (CIS) was diagnosed in 8 men (0.39%).
6 patients with CIS cells developed testicular tumors, one remained tumor-free and one was lost to follow-up.
|The findings are applicable to the selection of patients for biopsy and appropriate treatment of CIS when diagnosed.|
|Strader et al. ||Western Washington State, USA 1988||Population based case-control study||Patients diagnosed with TC between 1977 and 1983 (n = 333) and 675 healthy controls.|
Men with a history of cryptorchidism were 5.9 times more likely to develop TC than men without such history.
Men with unilateral cryptorchidism were at a greater risk of tumor development on the side of the nondescent testicle (relative risk of 8.0) than on the opposite side (relative risk of 1.6).
The risk tended to be smaller among cryptorchidic men who had undergone orchiopexy before adolescence.
|The study supports the hypothesis that one or more local factors may account for the increased risk of germ cell testicular tumors in cryptorchidic men.|
|Møller and Skakkebæk ||Denmark 1999||Population based case-control study||514 patients diagnosed with TC identified in the Danish Cancer Registry and 720 controls randomly selected from the Danish population.|
A reduced risk of TC associated with paternity (odds ratio of 0.63).
Patients who before TC had a lower number of children than expected, faced a relative risk of 1.98.
No corresponding protective effect associated with a higher number of children than expected was found.
Similar associations were recorded for seminoma and non-seminoma cases.
|Data supporting the hypothesis that compromised male fertility and TC share important etiologies.|
|Jacobsen et al. ||Denmark 2000||Cohort study||3530 Danish men, born between 1945–1980 and diagnosed with TC in the period of 1960–1993. Control: the total population of Danish men born between 1945–1980 (n = 1,488,957) and their biological children (n = 1,250,989).|
Men, who developed TC, had a reduced fertility prior to the diagnosis (odds ratio of 0.93).
A significantly lower proportion of boys was born to the patients when compared with the general population.
The reduction in fertility was more pronounced in men with non-seminoma.
The reduction in offspring sex ratio was independent of the TC type.
|The study confirms earlier results from less conclusive studies, and indicates that TC, subfertility and a female-biased sex ratio among newborns are interrelated by biological mechanisms.|
|Jacobsen et al. ||Denmark 2000||Cohort study||32,442 men who had a semen analysis done during 1963–1995.|
Patients with fertility issues were more likely to develop TC than other men (89 cases, incidence ratio of 1.6).
The risk was relatively constant with increasing time between semen analysis and cancer diagnosis.
Low semen concentration (incidence ratio of 2.3), poor spermatozoa motility (2.5), and high incidence of morphologically abnormal spermatozoa (3.0) were all associated with an increased risk of TC.
|The results emphasize on the existence of common etiologies for low semen quality and TC. Low semen quality may be associated with increased incidence of germ cell tumors.|
|Pasqualotto et al. ||Cleveland, USA 2003||Case study||Seven patients presenting with infertility, followed by eventual TC diagnosis over a 15-year period.|
Two men had elevated serum follicle stimulating hormone and luteinizing hormone levels, 1 an abnormally low serum testosterone level prior to the TC diagnosis. Tumor markers were normal in all patients.
The tumor was found on the right side in 4 patients and on the left in 3.
5 cases presented with a seminoma, 1 with Leydig cell tumor and 1 carcinoma in situ.
Follow-up on fertility status was available in 6 cases, only one patient established a pregnancy.
|Most of the men who have TC and male infertility will most likely present with a seminona. Men diagnosed with infertility should be thoroughly investigated to rule out diseases associated with their infertility.|
|Richiardi et al. ||Sweden 2004||Population based case-control study||4592 patients with TC and 12,254 control subjects.|
Before diagnosis, TC patients had lower number of children (odds ratio of 0.71), with a lower frequency of dizygotic twinning (odds ratio of 0.49).
Increased occurrence of twinning after diagnosis, probably due to treatment for iatrogenic infertility.
|The report provides evidence of an association between subfertility and the subsequent risk for TC.|
|Doria-Rose et al. ||Western Washington State, USA 2005||Case-control study||329 TC patients diagnosed from 1977 to 1983, and 672 cancer-free controls.||The results are consistent with an increased risk of TC among men with reduced fertility, going beyond the effects of cryptorchidism.|
|Walsh et al. ||State of California, USA 2009||Cohort study||A total of 51,461 couples evaluated for infertility from 1967 to 1998 linked with 22,562 TC patients.|
34 post-infertility-diagnosis cases of TC were identified.
Men seeking infertility treatment had an increased risk of subsequently developing TC (incidence ratio of 1.3), along with a markedly higher risk among those with known male factor infertility (odds ratio of 2.8).
|Men with male factor infertility have an increased risk of subsequently developing TC, suggesting common etiologic factors for infertility and TC.|
Infertility has been studied as a risk factor for PC development in the past. The most acknowledged report interrelating male infertility with prostate cancer incidence is a study by Walsh et al.
] focused on men treated in 15 clinics located in the state of California, US, and collecting data from 22,562 cases, 4549 of which had male factor infertility. The results showed that a larger proportion of men with male factor infertility developed prostate cancer compared with those without (1.2% vs.
0.4%) with an average time period from infertility evaluation to cancer diagnosis of 11 years. A total of 168 cases ultimately developed PC. This number was lower than the population standard, which was expected to reach 185 cases, suggesting that all patients in the cohort were at a lower risk for PC diagnosis. No significantly elevated chances for low grade PC occurrence was associated with patients having male factor infertility, while a 2-fold increase in the PC risks was recorded for high grade PC. Furthermore, men without male factor infertility were generally at a lower risk for PC when compared to the population, showing a 0.7 decrease in low grade PC, and a 0.8 decrease in high grade PC. When studying the duration of infertility treatment, men with male factor infertility were found to have 1.8 times the hazard of any PC development compared with those without male factor infertility, furthermore, they were 2.6 times more likely to develop high grade PC. An interesting finding was that for every year of male infertility treatment the patients were 1.2 times more likely to develop PC, meaning that after 5 years of treatment the patients were facing a 2-fold increase in the probability of PC development. Furthermore the study was able to conclude that each additional year to the age was accompanied by a 10% increased risk for PC development. Walsh et al
., admit that the results could be slightly biased, as men with male factor infertility were more likely to be screened for prostate cancer. They justify this conclusion by pointing out that the data did not cause substantial changes to the final result due to lack of significant differences in the low grade PC among men with male factor infertility [140
On the other hand, a variety of studies pointed out that reproductive dysfunction could in fact be associated with a lower risk of PC development in infertile men. Diverse experiments connecting the effects of androgens in cell growth and proliferation [141
] showed that decreased levels of androgens in animal models were associated with a decreased PC risk [143
], failing to prove that elevated androgen production could be associated with an increased risk for a prostate malignancy [144
]. This supports the idea of an androgen saturation model, according to which there is a point of a maximum threshold for androgens above which, the chances of PC development significantly decrease [145
]. One of the most influential and reliable studies elaborating on this hypothesis was conducted by Ruhayel et al.
] in Sweden, based on a large cohort of subjects according to the data obtained from the Swedish National Cancer Registry. The experimental design included 445 prostate cancer patients together with 446 controls and the hypothesis was based on the assumption that men with chronic testicular dysfunction would produce less androgens, thus face a decreased risk for a prostate malignancy. All childless men due to their free will or female factor infertility were excluded. Consistent with this hypothesis was the revelation of only two cases of non-fatal prostate cancer in an epidemiological study including 3518 men with the Klinefelter syndrome [8
]. This result was lower than average chances for a PC diagnosis and can be contributed to the hypogonadism that is commonly observed in these patients [47
]. Since a large number of infertile men had hypogonadism [96
] thus produced less androgens, their chances for PC development were significantly lower. The general conclusion of the study was that infertile men had half the risk for developing PC compared to men with proven fertility. Previous test trials have also supported this result, as Thompson et al.
] showed that patients treated with inhibitors of the testosterone conversion had a 25% lower incidence of a prostate tumor. Andriole et al.
] used a similar inhibitor and found a 23% lower probability of PC development. A study by Jørgensen et al.
] in Denmark used information gathered from the national population-based register, looking at the associations between infertility and PC. The team analyzed a pool of 3400 prostate cancer patients, concluding that childless men had a 16% (0.84) lower risk of PC development compared with men who had at least 1 child. A Swedish study by Giwercman et al.
] looked into the link between male testicular function and PC development. In an attempt to prove this hypothesis they conducted a nationwide population-based case-control study, through which they were able to identify all 48,910 PC patients born from 1916 onwards. The study hypothesized that childless men were less likely to develop PC when compared to men who have fathered children. The results showed a reduced risk of 0.83 in childless men compared to men with ≥2 children. The authors also believe that poor semen parameters are indicative of testicular dysfunction, which could be a protective measure against prostate cancer in the reproductive age. At the same time the authors acknowledge that their analysis of childless subjects alone may not represent an accurate measure for the effects of male factor infertility on eventual PC occurrence. Instead, they note that by reducing the category of childless men to patients with male factor infertility the results would indicate a further reduction of the risk for PC development. Furthermore, the study showed that the degree of fertility or infertility was associated with PC risks, as men with 0 children were facing a 0.83 odds ratio, men who fathered 1 child closely followed with a value of 0.93, and men with >2 children were facing a 1.0 ratio, defined as the reference value. Studies examining the relationship between prostate cancer and male infertility are summarized in Table 2
Epidemiologic studies of the association between male infertility and prostate cancer.
Epidemiologic studies of the association between male infertility and prostate cancer.
|Author(s)||Country and Year||Design||Subjects||Finding(s)||Conclusions|
|Giwercman et al. ||Sweden 2005||Population-based case-control||48,850 cases of PC between 1958–1998. For each case, one control was matched by year of birth.|
Men being childless or having fathered one child only were associated with reduced risks for PC compared to cases having fathered 2 or more children (odds ratio of 0.83 and 0.93; respectively).
There was no further change in risk associated with fathering of more than 2 children.
The risk for PC was reduced among childless men.
|A dysfunctional reproductive system supporting the prostatic growth to a lesser extent could be a feasible underlying cause of this association.|
|Negri et al. ||Italy 2006||Case-control study||1294 patients diagnosed with PC between 1991 and 2002, and 1451 controls as cases for a wide spectrum of acute and non-neoplastic conditions.|
Compared to men with 2 or more children, the odds ratio for childless men was 0.95 when adjusting only for age and geographic locality, and 1.10 after further adjustment for marital status and age at marriage.
The odds ratio was adjusted to 1.00 when unmarried and separated/divorced men were accounted for, 1.09 in terms of men below 65 years of age and 1.13 with respect to cases above the age of 65 years.
The odds ratio was 1.17 for men with only 1 child when compared to men who reported 2 or more children.
|The report concludes that the relation between the number of children and PC risk remains controversial.|
|Haralp et al. ||Israel 2007||Cohort study||15,268 fathers followed for 28–41 years from the birth of a live offspring.|
543 men with one or more stillborn offspring experienced an increased risk of PC (incidence ratio of 1.87).
With one reported stillbirth, the risk ratio was 1.68 and with two or more, the risk ratio was 3.29.
|The study suggests that stillbirth and PC may have shared environmental causes. Genetic susceptibility to PC might increase the risk of a stillbirth in offspring.|
|Jørgensen et al. ||Denmark 2008||Cohort study||All men born in Denmark between 1935 and 1988, among whom 3400 developed PC during follow-ups between 1968 and 2003.|
Childless men were at a 16% reduced risk of PC compared with fathers (incidence ratio of 0.84).
The sex of the offspring did not affect PC risk (odds ratio of 0.99).
Among fathers, a significant trend was observed of gradually reduced PC risk with the increasing number of children.
|Men without children are at a moderately reduced risk of PC. Among men with children, there appears to be a linear decline in PC occurrence with an increasing number of children, independent of the sex of the offspring.|
|Ruhayel et al. ||Sweden 2010||Case-control study||445 PC cases and 446 controls. 841 men were biological fathers and 50 men were infertile.||Infertile men were at a significantly lower risk of being diagnosed with PC than fertile men (odds ratio of 0.45).||Enduring male infertility may be associated with a reduced PC risk, validating the theory that normal testicular function and steroidogenesis are important factors to the later development of PC.|
|Walsh et al. ||State of California, USA 2010||Population-based case-control||A total of 22,562 patients being evaluated for infertility from 1967 to 1998, and linked to the cancer registry. The incidence of PC was compared with the incidence in an age- and geography-matched sample of men from the general population.|
168 cases developed PC development after infertility diagnosis.
Men evaluated for infertility but not specifically with male factors were not found to have an increased risk of cancer compared with the general population (incidence ratio of 0.9).
The highest risk was found in cases with male factor infertility who developed high–grade PC (incidence ratio of 2.0).
According to a multivariate analysis, men with male factor infertility were found to be 2.6 times more likely to be diagnosed with high–grade PC.
|Male infertility may be an early and identifiable risk factor for the development of clinically significant PC.|
|Wirén et al. ||Sweden 2013||Population-based case-control||117,328 PC cases and 562,644 controls, matched on birth year and residence.|
Childless men had a decreased risk of PC when compared to fathers (odds ratio of 0.83) and the risk was lower for low-risk PC (odds ratio of 0.74) than for metastatic PC (odds ratio of 0.93).
Adjustment for marital status and education narrowed the ratio in the low-risk category (0.87) whereas the odds ratio for metastatic cancer remained almost unchanged (0.92).
|The report claims that associations between the fatherhood status and PC are predominantly due to socioeconomic factors influencing health care-seeking behavior.|
5. Clinical Potential of MicroRNAs in the Diagnosis and Treatment of Male Infertility and Reproductive Cancers
Molecular biomarkers are a new and promising strategy to improve noninvasive diagnostics of male reproductive disorders, facilitating their management through effective screening, early diagnosis and more accurate prognosis. Furthermore these molecules may be more abundant in semen than in blood or urine, thus they may be more easily identified and quantified using PCR, RT-PCR or mass spectrometry. Biomarkers of male infertility, testicular or prostate cancer are now emerging, and it is indisputable that semen analysis through genomics and proteomics has the potential to complement other diagnostic tools available in urology and andrology clinics [154
MicroRNAs (miRNAs) are non-coding single-stranded RNA molecules of about 18–22 nt that play important roles in regulating posttranscriptional gene silencing via base pair binding to the 3' untranslated region of their target messenger RNAs (mRNAs) [155
]. Regulating the expression of more than 60% of genes responsible for protein encoding, miRNAs are involved in almost every biochemical process in the organism, hence their proper function is pivotal for a normal cellular development [157
]. Changes in the expression patterns of miRNAs could therefore affect gene transcription and/or translation, leading to a compromised spermatogenesis, or to the occurrence of several types of malignancies, including testicular or prostate cancer [157
Preliminary studies have reported that miRNAs such as miR-18a [159
], miR-122a [160
] and the miR-34 family [158
] are emerging as key players in germ cell function and cell fate determination, acting to interpret and transduce cellular signals in order to allow the maintenance of undifferentiated stem cell populations, while on the other hand allowing cell differentiation during spermatogenesis. These fundamental roles for miRNAs in germ cell development have implications for normal and disease states such as infertility and germ cell tumors [158
Testicular cancer has a unique miRNA expression profile, and several miRNA molecules have been implicated in its neoplastic development, e.g., miR-372 and miR-373 [162
]. The first report suggesting interactions between male infertility and TC via miRNAs was published by Voorhoeve et al.
]. According to this study small RNAs derived from novel oncogenes Mirn322 and Mirn323 could mediate the expression of mRNAs derived from these genes, hence they could play a role in developing testicular germ cell tumors [162
]. Subsequently a high-throughput microRNAome analysis in human germ cell tumors revealed that the expression profiles of 156 miRNAs differed in type II and type III TC subjects suggesting the importance of miRNA in male infertility due to a testicular malignancy in some cases [163
]. A novel molecular connection between male infertility and TC has been proposed via miR-383 regulation [164
]. miR-383 expression has been shown to be downregulated in the testes of infertile men with maturation arrest. At the same time, downregulation of this small RNA results in enhanced proliferative activity of germ cells. While a direct target of miR-383 is the interferon regulatory factor-1 (IRF1), which has been identified as a tumor-suppressor gene, it seems to have a pro-mitogenic role in spermatogonia and early spermatocytes. The inhibition of IRF1 as a result of miR-383 activity leads to reduced levels of signaling molecules exhibiting antiproliferative and tumor-suppressor effects. Thus, disruptions of the miR-383 expression may lead to spermatogenic failure as well as promotion of testicular carcinoma cell proliferation [165
To date, most of the studies related to the roles of microRNAs in prostate cancer highlight the potential of miR-141, miR-200b and miR-375 as significant disease correlates, which could potentially be used in tests at the time of PC diagnosis [166
]. It is important to note that there appears to be a complex interaction between androgen signaling in PC, microRNA expression, and various key pathways in prostate tumorigenesis. In essence, certain miRNAs have been shown to be regulated by androgen-receptor (AR) mediated signaling while others are involved in modulating the function of the AR signaling pathway, providing additional evidence to an intricate endocrine interplay in the male reproductive system [167
]. MiR-125b has emerged as a prominent androgen-responsive microRNA molecule whose upregulation may result in androgen-independent growth of prostate tumors, most likely through its anti-apoptotic effects [168
]. A substantial interest has risen with respect to the miR-34 family—a group of putative tumor suppressors, which were originally found to be a direct target of p53. According to Cheng et al.
] suppression of these molecules leads to an expansion of the prostate stem cell compartment and the development of early invasive and high-grade prostatic intraepithelial neoplasias. Consistently, a combined deficiency of p53 and miR-34 resulted in an acceleration of self-renewal, and motility of prostate stem/progenitor cells, providing a direct genetic evidence emphasizing that miR-34 deserves further examinations with respect to its roles as a key component of prostate stem cell compartment regulation, aberrations of which may lead to cancer occurrence [169
]. In the meantime, miR-18a, related to the occurrence of testicular cancer, has been shown to be upregulated in clinical prostate tumor specimens and cancer cell lines as well. miR-18a knockdown decreased cell growth in PC cells, and significantly reduced prostate tumorigenesis in in vivo
nude mice through apoptotic mechanisms, thus it may represent a therapeutically appealing option for PC treatment [170
Given the redundancy of miRNAs, there is a strong prospect for miRNAs to be involved in driving and coordinating the expression of hallmark characteristics related to altered spermatogenesis and reproductive cancers. Therefore, understanding the role of various miRNAs at different stages of a pathological growth, along with their individual expression patterns, may provide vital information to the search for prognostic biomarkers and discover potential therapeutic targets [167
]. There are essentially two strategies by which miRNA molecules could be targeted to treat diseases—either through a reduction of the expression or effects of a specific miRNA, or through the induction of a pathology-suppressing miRNAs within affected cells [171
]. Nevertheless, prior to taking advantage of the miRNA potential in male infertility or cancer treatment, several issues must be overcome. One of the major obstacles is the delivery and significant expense of artificially modified nucleic acids. While frequent injections could be a possible treatment option for oncological cases, this is unlikely to be used in subfertile but otherwise healthy individuals [157
]. Targeting of miRNA mimics and anti-MiRs remains another limiting factor in the viability of miRNA treatment, primarily due to the fact that miRNA molecules have a multitude of functions within various tissues. Meanwhile, the use of specific lipid or polymer-based nanoparticles, adenoviral or lentiviral vectors to deliver miRNA mimics has provided promising results in a successful delivery and long-term expression within specific cell types [171
]. Summarizing, as the miRNA field advances, new methods of studying specific characteristics and behavior of microRNAs in a biological system, coupled with recent biomedical progress achieved with therapeutic miRNAs using nanotechnologies is encouraging and it is expected to initiate a real clinical development of therapeutic miRNAs soon [172