The use of fluorescent in situ hybridization in male infertility

Abstract

Male factors are implicated in up to 50% of couples being evaluated and treated for infertility with advanced assisted reproductive technologies. Genetic abnormalities, including sperm chromosome aneuploidy as well as structural aberrations, are one of the major causes of infertility. The use of chromosome-specific DNA probes labeled with fluorochromes, particularly the combination with multiple probes, has been used to indirectly study the sperm chromosome by fluorescent in situ hybridization (FISH). Clinically, this technique is also used to assess the sperm of men recovering from gonadotoxic treatment. Recent advances in this technology facilitate the evaluation of sperm aneuploidy. Sperm FISH is a widely used screening tool to aid in counseling couples with severe male factor infertility, especially in cases of prior repeated in vitro fertilization/intracytoplasmic sperm injection failure or recurrent pregnancy loss. Automation of FISH imaging and analysis, as well as the development of emerging techniques such as comparative genomic hybridization, will all contribute to the promise of future diagnostic approaches aimed at improving the quality, ease, and efficiency of aneuploidy analysis.

Introduction

Infertility is defined as the inability to conceive despite trying for a year and it affects approximately 12% of the reproductive age population, and roughly one-third of these cases can be attributed to a male factor [Chandra et al. 2005]. It is considered a genetic lethal as the family lineage stops at that individual with no progeny produced. A genetic defect associated with that individual cannot be transmitted to the offspring ensuring the maintenance of reproductive fitness of the species. However, with the advent of assisted reproductive technologies we can overcome many types of sterility and bypass nature’s protective mechanisms that were developed in evolution to prevent fertilization by defective or deficient sperm. In the past, treatment of male infertility with in vitro fertilization (IVF) was largely unsuccessful; yet the development of intracytoplasmic sperm injection (ICSI) has revolutionized the treatment of severe male infertility, allowing many men the opportunity to father their own children [Palermo et al. 1992].

The causes of male factor infertility are multifactorial with an estimated 50% due to or contributed by genetic abnormalities [Lipshultz and Lamb, 2007]. There are many well-documented genetic causes of infertility, such as cystic fibrosis (reviewed in Matzuk and Lamb [2008]). Although, with the current limits of diagnostic testing and in the absence of a thorough clinical evaluation, many genetic etiologies go undiagnosed. While fertilization in cases of severe spermatogenesis defects is facilitated with the advent of ICSI, there is an increased risk of sex chromosome aneuploidies (and birth defects) in the offspring of these men [Alukal and Lamb, 2008; Hansen et al. 2005; Ludwig, 2005; Rimm et al. 2004]. The long-term implications for offspring conceived through ICSI remain largely unknown.

Genetic studies define a relationship between sperm abnormalities and meiotic errors [Sarrate et al. 2005]. These errors are associated with mutations in meiosis-specific genes, such as those involved in the processes of DNA recombination and repair [Baarends et al. 2001]. Environmental factors are also hypothesized to result in meiotic deficiencies [Mroz et al. 1999]. Abnormal meiotic recombination, which causes the production of aneuploid gametes, may also give rise to different degrees of meiotic arrest many times occurring at specific stages of meiosis [Martin, 2006; Egozcue et al. 2005].

The routine semen analysis, measuring sperm concentration, motility, morphology, and the presence of other cells, is one of the first tests ordered in the evaluation of the infertile male. Indicators of patency and function of the male genital tract are assessed including volume, liquefaction time, pH, and the presence or absence of fructose. The routine semen analysis only provides clues that may indicate the need to perform additional evaluation. The incidence of chromosomal aberrations may be increased in sperm from men with an abnormal semen analysis [Burrello et al. 2005; Machev et al. 2005; Sarrate et al. 2005]. Indeed, gametes of infertile men show a higher rate of chromosome abnormalities than the general population [Egozcue et al. 2003]. Nevertheless, the selection of normal morphology sperm does not necessarily indicate the sperm has the normal haploid chromosomal complement [Ryu et al. 2001].

As new technologies enter the clinical arena, our knowledge of spermatogenic deficiencies resulting in abnormal cytogenetic analysis of human sperm increased. Fluorescent in situ hybridization (FISH) facilitated the detection of sperm aneuploidy. Spermatozoa consist of the head with a nucleus containing the highly compacted male haploid genome, and the flagellum, which is responsible for sperm motility. Sperm chromatin is a highly organized, compact structure consisting of DNA and heterogeneous nucleoproteins, which are essential for the accurate transmission of genetic information to offspring. Sperm FISH is most commonly used to determine the proportion of aneuploidy present in sex chromosomes and autosomes of infertile men. Furthermore, it can also quantify the probability of transmitting aneuploidies and complex chromosomal rearrangements, such as translocations and inversions [Sarrate et al. 2005]. As a result, sperm FISH analysis is increasingly included in infertility diagnostic protocols, providing useful information to enable the affected couple to make difficult, but informed reproductive choices.

History of FISH

In 1978, Rudak and coworkers published the first account of the evaluation of sperm chromosome abnormalities by modifying the human sperm–hamster oocyte penetration assay system previously developed by Yanagimachi and colleagues in 1976 [Rudak et al. 1978; Yanagimachi et al. 1976]. The method and protocol were then later standardized by Martin et al. [1982]. Hamster eggs are ‘promiscuous’ allowing heterologous sperm to penetrate and undergo decondensation, although fertilization does not proceed further [Yanagimachi et al. 1976]. This fusion system allows precise karyotypes to be obtained in which numerical and structural abnormalities can be assessed for each chromosome. However, this procedure is technically difficult and time consuming, with low yield of analyzable cells, and costly. In addition, sperm capacitate and penetrate zona-free oocytes, which precludes the analysis of sperm from men with sperm function deficiencies or severe oligospermia [Martin, 2007].

Despite these technical difficulties, several research laboratories established a fairly consistent estimate of structural or numerical abnormalities of sperm in normal fertile men [Templado et al. 1996; Brandriff et al. 1985; Martin et al. 1983]. In these men the rate of aneuploidy was generally less than 2%. These studies laid the foundation for the development of advanced techniques to study sperm aneuploidy. Chromosome-specific DNA probes were initially labeled with radioactive compounds to permit detection of the hybridized probe [Joseph et al. 1984] and later with fluorochromes [Pieters et al. 1990] that allowed simultaneous detection of multiple chromosomes [Wyrobek et al., 1992].

Chromosome-specific DNA probes for FISH were developed in the 1990s to evaluate sperm chromosome aneuploidy [Holmes and Martin, 1993]. Sperm function deficiencies do not affect FISH analysis [Egozcue et al. 1997; Robbins et al. 1997]. Sperm FISH defines the frequencies of chromosomal aberrations present in an ejaculate, routinely measuring chromosomes X, Y, 13, 18 and 21. These chromosomes are analyzed because they are consistent with a viable offspring with sex chromosome aneuploidies (such as Klinefelter’s syndrome or Turner syndrome) or Patu’s, Edward’s or Trisomy 21 (Down’s syndrome).

Technical aspects

Analysis of sperm chromosomes with the human–hamster ova system was limited because multiple experiments were required to obtain a small number of metaphase spreads. In contrast, multicolor FISH is performed in less time, allows the analysis of thousands of sperm increasing the statistical power of the analysis and, with the use of sophisticated image analysis software, does not require a skilled cytogeneticist to analyze the results. The limitations of FISH include the inability to study structural anomalies as well as the limited evaluation as only the segments of chromosome identified by the probes are detected. This limitation contrasts with the human–hamster fusion system, where the entire chromosome complement is studied in just a few representative cells.

The highly compacted chromatin in the sperm head requires additional decondensation treatment to make the DNA sequences accessible to the probes used in FISH. Reportedly, dithiothreitol (DTT), a reducing agent that breaks the disulfide bonds found in the DNA chains, currently yields the best and most reliable results [Martin and Rademaker, 1995; Wyrobek et al. 1990]. Analysis of 1×10⁴ spermatozoa per individual and per set of probes is recommended [Martin et al. 1996; Moosani et al. 1995]. An internal autosomal control probe is needed to differentiate between disomic and diploid spermatozoa.

Chromosome aneuploidy scoring is based on the number and characteristics of the signals observed. A strict criterion for this scoring eliminates subjective variation. These criteria include: (1) overlapped spermatozoa or sperm heads without a well-defined boundary are not counted; (2) in cases of disomy or diploidy, all signals should have the same intensity and be separated from each other by a distance longer than the diameter of each signal; and, finally, (3) nullisomies are not directly scored and are conservatively considered as equivalent to the incidence of disomies [Blanco et al. 1996].

When all of the technical considerations are taken into consideration, the use of sperm FISH allows an accurate analysis of the incidence of chromosomal aberrations for the diagnosis of infertile men.

Clinical uses of FISH in male infertility

An adjunct to conventional cytogenetic analysis

Cytogenetic analysis by karyotype of peripheral blood lymphocytes and polymerase chain reaction (PCR) testing for Y-chromosome microdeletions are the genetic tests currently recommended by the American Urological Association (AUA) and American Society of Reproductive Medicine (ASRM) to detect genetic abnormalities in men with a sperm density ≤ 5 × 10⁶ who are considering IVF [American Urological Association and American Society of Reproductive Medicine, 2001]. Since the 1990s, FISH has been used as an adjunct to karyotype to confirm or further delineate cytogenetic abnormalities. In male infertility, FISH testing is most often applied to men with structural sex chromosome abnormalities, particularly in the case of the XX phenotypic male. At our institution, phenotypic males with an XX karyotype routinely undergo FISH analysis to determine whether the sex-determining (SRY) region is present (Figure 1). Additional probes, such as those specific for the Xpter (XPAR1) pseudoautosomal regions, were developed to detect other regions of the Y chromosome for the diagnosis of XX SRY negative males [Rigola et al. 2002]. Some Y-chromosome microdeletions, rearrangements, duplications, and inversions may be better characterized using FISH, when used in combination with the routine karyotype and multiplex PCR [Pinho et al. 2005; Valetto et al. 2004].

Figure 1.

Fluorescent in situ hybridization (FISH) analysis of all 46 chromosomes in an XX phenotypic male. The patient stained positive (red) for the SRY region at the tail of one of the X chromosomes.

FISH of peripheral white blood cells is used to determine mosaicism, in addition to karyotype methods that employ conventional Giemsa (G-)banding. In a study of men with Klinefelter’s syndrome, Abdelmoula and colleagues demonstrated that FISH easily and accurately identified men with mosaic Klinefelter’s syndrome [Abdelmoula et al. 2004]. The authors concluded that FISH is more specific and more sensitive for detecting mosaicism, and recommended that FISH analysis should be used in all suspected cases of XXY mosaicism. In a much higher population of newborn children screened by cytogenetic analysis, a large number of children found to have <10% mosaicism using conventional G- or R-banding were proven to be nonmosaics by FISH [Nielsen and Wohlert, 1991]. Therefore, FISH is recommended to confirm mosaicism if less than 10% of cells are mosaic by karyotype.

Sperm FISH

Human sperm FISH was introduced to determine aneuploidy in the sperm of oligospermic and aging men [Martin, 2007; Miharu et al. 1994; Holmes and Martin, 1993], and over the past decade, the technology has improved. Sperm FISH is most commonly used to determine the proportion of aneuploidy present in sex chromosomes and autosomes of infertile men (Figure 2).

Figure 2.

Examples of sperm fluorescent in situ hybridization (FISH). Top left: normal X- and Y-bearing sperm. Top right: sperm with chromosome 18 and XY disomy. Bottom left: sperm with sex chromosome XY disomy. Bottom right: sperm with chromosome 13 and 18 disomy.

Both retrospective [Carrell et al. 2001; Blanco et al. 1998] and prospective studies [Nagvenkar et al. 2005] show that increased sperm aneuploidy measured by sperm FISH, increases the risk of IVF/ICSI failure and fetal aneuploidy. In addition, testicular sperm from men with nonobstructive azoospermia (NOA) displays higher rates of aneuploidy in spermatozoa and blastomeres in IVF/ICSI-derived embryos [Gianaroli et al. 2005]. Palermo and colleagues demonstrated that testicular sperm from men with NOA had a higher rate of aneuploidy than epididymal sperm from men with obstructive azoospermia (OA) [Palermo et al. 2002]. These studies have helped validate the prognostic value of aneuploidy testing by sperm FISH in male infertility.

Sperm FISH can define aneuploidy in human epididymal and testicular sperm. In 1999, Huang and colleagues first noted an increased incidence of sex-chromosome aneuploidy in men with NOA (39–45%) compared with normal controls (29%) [Huang et al. 1999]. Another study demonstrated that epididymal sperm from men with OA had a higher rate of aneuploidy than ejaculated sperm from fertile men, although this did not lead to differences in IVF/ICSI success [Sukcharoen et al. 2003]. This has led to the recommendation of considering FISH in testicular or epididymal sperm samples especially after repeated IVF/ICSI failure.

Sperm FISH defines the proportion of aneuploidy present in the ejaculates of men with a variety of genetic conditions and clinical syndromes (Table 1). Sperm morphology cannot be used to predict genetically normal sperm [Ryu et al. 2001]. Conversely, men with severe sperm morphologic abnormalities have higher rates of aneuploidy including tail agenesis [Carrell et al. 2004], double-headed sperm [Lewis-Jones et al. 2003], round head-only syndrome or globozoospermia [Morel et al. 2004], necrozoospermia, and multiple flagella [Devillard et al. 2002]. Collodel and colleagues demonstrated that men with isolated asthenospermia, especially with sperm motility under 30%, had a much higher rate of sperm disomy and diploidy [Collodel et al. 2007a]. Increased rates of sex chromosome aneuploidy have also been described in patients with Y-chromosome microdeletions and oligospermia [Minor et al. 2007], as well as those with Klinefelter’s syndrome, display an increased risk of aneuploidy in both ejaculated and testicular sperm. Interestingly, normospermic men who are partners in couples with unexplained recurrent miscarriages and repeated IVF failure have a higher rate of sperm aneuploidy [Petit et al. 2005; Bernardini et al. 2004].

Table 1.

Indications for sperm fluorescent in situ hybridization (FISH).

Indication	Study
Recurrent pregnancy loss	Bernardini et al. [2004]
Repeated in vitro fertilization failure	Petit et al. [2005]
Oligospermia	Martin [2007]
Low motility <30% (asthenospermia)	Collodel et al. [2007a]
Nonobstructive azoospermia (testicular sperm)	Palermo et al. [2002]
Obstructive azoospermia (epididymal sperm)	Sukcharoen et al. [2003]
Teratozoospermia
Tail agenesis	Carrell et al. [2004]
Multiflagellar sperm	Devillard et al. [2002]
Macrocephallic sperm	Devillard et al. [2002]
Round head-only syndrome (globozoospermia)	Morel et al. [2004]
Necrozoospermia	Moretti et al. [2007]
Double-head or multinucleated sperm	Lewis-Jones et al. [2003]
Other severe morphologic defects	Carrell [2008]
Genetic abnormalities
Robertsonian translocations	Sarrate et al. [2005]
Reciprocal translocations	Sarrate et al. [2005]
Chromosome inversions	Sarrate et al. [2005]
Ring chromosome	Arnedo et al. [2005]
Klinefelter’s syndrome (mosaic and nonmosaic)	Kruse et al. [1998]
	Lim et al. [1999]
	Rives et al. [2000]
Y-chromosome microdeletions	Minor et al. [2007]

In 2005, Sarrate and colleagues concluded that men with heterozygous pericentric inversions or Robertsonian translocations tend to produce sperm with normal, balanced chromosomes (55.9–99.3% and 72.2–92.8%, respectively) [Sarrate et al. 2005]. However, carriers of heterozygous reciprocal translocations varied in the proportion of unbalanced sperm (29.4–70.2%). Indeed, a patient with a ring Y chromosome with significantly increased rates of sex chromosome and autosomal disomy and diploidy naturally fathered a son with Klinefelter’s syndrome [Arnedo et al. 2005].

From this discussion, one can conclude that men with a number of different male infertility diagnoses have increased rates of aneuploidy compared with fertile men. However, diagnosis using sperm FISH is not frequently performed in the United States because of the high cost of the reagents, as this test is also rarely covered by medical insurance. From a practical perspective, sperm-FISH analysis is most important for couples with recurrent miscarriages or IVF (with or without ICSI) failures to determine whether sperm aneuploidy is present. Sperm-FISH testing is also highly informative for patients with severe teratoozoospermia with oligospermia, as well as oligoasthenoteratozoospermia. Sperm-FISH analysis defines a meiotic defect and this knowledge can be used to help direct reproductive counseling, clinical management, and to allow the couple to make an informed reproductive decision to perhaps chose preimplantation genetic diagnosis (PGD) testing should they elect IVF and/or prenatal genetic testing, adoption or donor sperm. Couples with abnormal sperm-FISH should always be referred to a genetic counselor to further understand the risks, benefits, and their alternatives in family planning.

PGD during IVF and prenatal diagnosis

PGD employs either FISH or PCR to detect specific genetic defects along with numerical and structural chromosomal abnormalities in a fertilized embryo. Embryos derived from the sperm of men with genetic disorders such as Klinefelter’s syndrome [Staessen et al. 2003], Y-chromosome deletions [Mansour, 2004], Robertsonian translocations [Keymolen et al. 2009], and complex chromosomal rearrangements [Lim et al. 2008] undergoing IVF/ICSI can be biopsied and tested for their specific disorder in an attempt to transfer and implant unaffected embryos. Men with Y-chromosome microdeletions undergoing IVF/ICSI can select XX embryos in an attempt to avoid transmission of their genetic defect [Stouffs et al. 2005]. Couples suffering from recurrent miscarriages or IVF failure can also be offered PGD to help select embryos without aneuploidy or other genetic disorders [Rubio et al. 2009].

Chorionic villus sampling and amniocentesis can provide tissue for prenatal detection of certain genetic disorders using FISH in a similar manner to PGD. Multicolor FISH can be performed by labeling all 24 chromosomes with different color fluorescent probes, which enhances the ability to detect chromosomal abnormalities [Cetin et al. 2005]. When used in combination with conventional karyotype, FISH will determine whether a fetus carries a particular disorder such as Y-chromosome microdeletion and rearrangements [Toth et al. 2001]. Prenatal diagnosis can help a couple decide whether to terminate a pregnancy, facilitate genetic counseling, and prepare a family for the birth of an affected child.

Chromosomal aberrations defined after testicular injury

Rodent models

Rodent models of testicular injury provided unique approaches to assess numerical chromosomal aberrations by multicolor sperm FISH. In 1995, a mouse testicular sperm aneuploidy assay was developed to detect aneuploidy in sex and autosomal chromosomes in mouse spermatozoa [Wyrobek et al. 1995] and later expanded to assess epididymal sperm [Lowe et al. 1996]. Eventually, the same group developed a similar multicolor sperm-FISH assay in rats [Lowe et al. 1998].

Rodent sperm FISH can define numerical chromosomal aberrations after the administration of potential gonadotoxins. Administration of chemotherapy agents, such as etoposide and merbarone, induced disomy and diploidy [Attia et al. 2002], while other drugs such as taxol [Adler et al. 2002] and vinblastine [Schmid et al. 2001] caused either borderline significant increases in disomic sperm or inconsistent increases in disomic sperm, respectively. More recently, amsacrine and nocodazole were similarly shown to increase sperm disomy [Attia et al. 2008]. In the rat model, chronic low-dose cyclophosphamide significantly increased autosome disomy or nullisomy, but did not increase Y chromosome disomy or diploidy [Barton et al. 2003].

Mouse sperm FISH also detects chromosomal abnormalities after the administration of pharmaceuticals. Diazepam, an anxiolytic [Schmid et al. 1999], and griseofulvin, an antifungal [Shi et al. 1999], increase both disomy and diploidy in a dose-dependent manner. Thiabendazole, an antihelminthic agent, caused an increase in sperm diploidy, while colchicine, an anti-inflammatory medicine commonly used for gout, induced higher frequencies of disomic sperm. Conversely, albendazole, another antihelmenthic agent, did not [Schmid et al. 1999].

The role of pesticides in the induction of sperm aneuploidy provided insight into the actions of these agents during meiosis. Carbendazim induced sperm diploidy in mice [Adler et al. 2002] and rats [de Stoppelaar et al. 1999]. Initially, acrylamide, a chemical used in water treatment and found in tobacco smoke, was reported to not cause chromosomal aberrations [Schmid et al. 1999], but later an increase in chromosomal mosaicism was found in two-cell embryos after male mice were given acrylamide [Marchetti et al. 2009].

Sperm FISH in rodents can be used to identify gonadotoxins and unsafe pharmaceutical agents during product safety testing.

Exposures to gonadotoxins in humans and sperm aneuploidy

Shortly after the development of sperm-FISH methodologies, investigators studied the effects of different pharmaceuticals, antioxidants, occupational exposures, smoking and pesticides on sperm aneuploidy [Wyrobek et al. 2005] (Table 2). Despite inconsistent results, combined data from each study shows a significant increase in sex chromosome and autosome aneuploidy after gonadotoxin exposure [Robbins et al. 2005]. While controlling for smoking and alcohol consumption, Robbins and colleagues found an increase in sex chromosome disomy and diploidy with high levels of caffeine intake [Robbins et al. 1997], although Rubes and colleagues did not confirm this finding [Rubes et al. 1998]. In a study of the deleterious effects of alcohol on sperm aneuploidy, researchers reported an association between increased alcohol consumption and sex chromosome disomy [Robbins et al. 1997]. Nevertheless, the potential confounding effects of smoking, caffeine, and alcohol intake in each of these articles makes drawing any broad conclusions difficult without rigorously controlled prospective studies. There is less known about the effects of nutrition on meiotic competency. In one study of the effects of dietary folate, zinc, and antioxidants on sperm aneuploidy, the authors conclude that only folate was associated with a decrease in sperm aneuploidy [Young et al. 2008].

Table 2.

Impact of exposure to gonadotoxins on sperm aneuploidy.

Chemical	Sex chromosome disomy	Autosome disomy	Diploidy	Study
Lifestyle factors
Caffeine	Increased	Increased	Increased	Robbins et al. [1997]
Alcohol	No change	No change	Increased	Robbins et al. [1997]
Smoking	Increased	Increased	No change	Robbins et al. [1997], Rubes et al. [1998]
Pharmaceuticals
Diazepam	Increased	Increased	No change	Baumgartner et al. [2001]
Finasteride	Increased	No change	Increased	Collodel et al. [2007b]
FSH	Decreased	No change	Decreased	Piomboni et al. [2009]
Vitamins and minerals
Folate	Decreased	Decreased	Decreased	Young et al. [2008]
Zinc	No change	No change	No change	Young et al. [2008]
Vitamin C	No change	No change	No change	Young et al. [2008]
Vitamin E	No change	No change	No change	Young et al. [2008]
β-carotene	No change	No change	No change	Young et al. [2008]
Antioxidant composite	No change	No change	No change	Young et al. [2008]
Chemotherapy
NOVP	Increased	Increased	Increased	Frias et al. [2003]
MACOP-B	No change	No change	No change	Martin and Rademaker [1995]
ABVD	Increased	Increased	Increased	Tempest et al. [2008]
BEP	Increased	Increased	Increased	Martin et al. [1999], De Mas et al. [2001]
Occupational exposures
Organophosphates	Increased	No change	No change	Padungtod et al. [1999]
Miscellaneous pesticides	No change	No change	No change	Smith et al. [2004]
Styrene	No change	No change	No change	Naccarati et al. [2003]
Acrylonitrile	Increased	Increased	NA	Xu et al. [2003]

FSH, follicle-stimulating hormone; NOVP, novantrone, oncovin, velban, and prednisone; MACOP-B, methotrexate, doxorubicin, cyclophosphamide, vincristine, bleomycin and prednisone; ABVD, bleomycin, doxorubicin, vincristine, and dacarbazine; BEP, bleomycin, etoposide, and cisplatin; NA, not applicable.

Sperm aneuploidy can occur following chemotherapy. Bleomycin, etoposide, and cisplatin (BEP) administration to patients treated for testicular cancer elicited an increased incidence of sperm aneuploidy that typically returned to baseline within 2 years after treatment [De Mas et al. 2001; Martin et al. 1999]. Similarly, patients treated for Hodgkin’s lymphoma receiving bleomycin, doxorubicin, vincristine, and dacarbazine (ABVD) or novantrone, oncovin, velban, and prednisone (NOVP) had an increased incidence of sperm disomy and diploidy after therapy, but this aneuploidy typically returned to baseline levels months after treatment [Tempest et al. 2008].

Baumgartner and colleagues described two men, who had chronically ingested diazepam (>0.3 mg/kg/day) for more than 6 months, who displayed a significant increase in sperm aneuploidy [Baumgartner et al. 2001]. Administration of finasteride was associated with increased sperm diploidy and sex chromosome disomy, which did not improve 1 year after cessation of the drug [Collodel et al. 2007b]. Reportedly, follicle-stimulating hormone administration significantly reduced sperm aneuploidy along with improving functional and structural sperm characteristics in oligospermic men, although the mechanism of action is not clearly understood [Piomboni et al. 2009].

Occupational exposure of male agricultural workers to organophosphate pesticides was associated with an increased frequency of sperm aneuploidy and sex chromosome disomy [Recio et al. 2001; Padungtod et al. 1999]. Furthermore, after exposure to the known gonadotoxin benzene, an increase in sperm disomy and diploidy was observed [Liu et al. 2003; Li et al. 2001]. Thus, heterogeneity of spermatogenic response to different gonadotoxins and occupational exposure to chemicals is possible.

Sperm aneuploidy may be associated with two very common infertility-related defects, cryptorchidism and varicoceles. Men with unilateral or bilateral cryptorchidism displayed a nonsignificant increase in disomy and diploidy even after orchidopexy [Moretti et al. 2007]. Similarly, varicoceles can increase sperm aneuploidy. Baccetti and colleagues reported that 31/38 infertile men with clinical varicoceles had a higher frequency of sperm, disomy or diploidy in their ejaculate [Baccetti et al. 2006].

The future of FISH

Recent advances have been made in the diagnostic use of FISH in male infertility, and are under investigation to improve future applications. Two major disadvantages of current FISH technology are the high cost and the amount of manual labor needed to analyze the thousands of sperm. The current cost of multicolor sperm FISH approaches US$1000. Individual probes cost approximately US$100 each, and typically two or three rounds of FISH are necessary. In our experience, manual microscopic FISH analysis is very laborious typically requiring 5–10 hours to count thousands of sperm. In 2002, Siffroi and colleagues proposed using filters to collect sperm in cases of extreme oligospermia before performing FISH [Siffroi et al. 2002], but other laboratories have not adopted this.

Carrell and Perry and colleagues first reported the use of automated sperm FISH, demonstrating results comparable to manual sperm FISH [Carrell, 2008; Perry et al. 2007]. Despite the high cost of the automated machinery (minimum of US$100,000), they described three advantages: (1) greatly reduced technician time to as little as 1 hour; (2) improved storage of images and data from each sample; (3) automated cell search abilities allowing manual analysis of samples with low levels of sperm [Carrell and Emery, 2008]. Stored images from each sample may allow subsequent manual analysis if necessary. Automated FISH analysis improves cost, as a direct outcome of reduced technician time, as well as precision and accuracy as the technology improves.

Primed in situ (PRINS) fluorescence hybridization and peptide nucleic acid (PNA) hybridization represent possible alternatives to conventional sperm FISH. PRINS employs unlabeled DNA probes that are hybridized to sperm DNA in the presence of primer-dependent DNA polymerase that synthesizes DNA in the presence of labeled nucleotides. The advantage of the PRINS techniques is that it is faster and cheaper to design custom oligonucleotide probes. The PRINS procedure was first introduced to human sperm by Pellestor and colleagues in 1995, but poor initial accuracy of the assay hampered its use [Pellestor et al. 1995]. PNA hybridization uses synthetic DNA mimics based on a peptide backbone. In 2003, Pellestor and colleagues first reported the use of PNA in human sperm [Pellestor et al. 2003]. The authors subsequently have improved the efficiency and specificity of PRINS and PNA hybridization [Pellestor, 2006], although its use in human sperm has not been widely adopted and conventional FISH remains mainstream.

Other alternatives to sperm FISH to analyze aneuploidy are quantitative fluorescence (QF)-PCR and array comparative genomic hybridization (aCGH). QF-PCR uses multiplex PCR with fluorescent primers to analyze fluorescence intensities in order to determine aneuploidy. QF-PCR has the potential to be less costly and time intensive [Carrell, 2008]. aCGH or microarrays have the potential to detect sperm aneuploidy and submicroscopic copy number variations throughout the genome. With the potential to analyze greater than 1,000,000 foci, aCGH can greatly enhance the accuracy of aneuploidy analysis. In male infertility, aCGH has also been proposed as an alternative to PCR to detect Y-chromosome microdeletions [Osborne et al. 2007]. The main disadvantage of aCGH is the cost (US$500–1000). Also, to determine sperm aneuploidy, individual sperm would need to be extracted and analyzed separately. Experience with applying QF-PCR or aCGH to sperm DNA has not been published to date.

Conclusions

Since its recent introduction, FISH has been heavily incorporated into the evaluation and treatment of the infertile male. While basic sperm FISH is relatively simple and can be performed by most cytogenetic and reproductive laboratories, multicolor FISH is a complex test requiring highly skilled and trained technicians. Disadvantages such as cost, technician time, and the limited number of chromosomes examined may be alleviated with the development of automated FISH. Sperm FISH can determine the rate of sex chromosome and autosome aneuploidy in ejaculated, epididymal, and testicular sperm, and can be useful in a wide spectrum of male infertility diagnoses. Information gathered from sperm FISH can enhance reproductive counseling, guide clinical management in family planning, and determine the need for PGD. Researchers can also use sperm FISH to further evaluate gonadotoxicity of existing or developing pharmaceuticals and suspected gonadotoxins. Further studies are needed to determine other potential indications in male infertility, cost effectiveness, and to establish clinical guidelines that incorporate FISH or other methods of testing for chromosomal aneuploidy.