Medical Device Link.

IVD Technology Magazine
IVDT Article Index

The genetic bases of male infertility

Marijo Kent-First, Angela Ryan, Richard Schifreen, and Susan Frackman

Although the human desire to have children is deeply ingrained, as many as 14% of all couples are affected by infertility. Of these, approximately 40% of the problems relate to the female, 40% to the male, and 20% relate to combined problems.

Infertility and subfertility issues are accentuated when partners wait until later in life before attempting to have children. Once a problem is suspected, both partners must undergo evaluation. The initial patient work-up for suspected male-factor infertility involves sperm analysis and hormone testing. Sperm analysis may show azoospermia (the absence of sperm in the ejaculate), oligozoospermia (fewer than 20 million sperm per ml), or normal sperm counts with abnormal morphology. More complex testing may include karyotyping.

In some cases testing reveals an obvious genetic etiology such as an XX male (with a Y to X translocation) or even a 47 XXY male (Klinefelter's syndrome). In such patients the testes are usually devoid of germ cells. In most cases, if initial hormone replacement therapy fails to improve sperm parameters, the patient must be referred for assisted reproductive technology (ART).

Until the advent of intracytoplasmic sperm injection (ICSI), severe male-factor infertility was largely untreatable. ICSI involves direct injection of sperm or spermatids into the egg. The procedure was quickly applied to human ART with limited studies in nonhuman species. ICSI has allowed infertile men to father children.

Although many apparently healthy children have been derived from ICSI, reports of chromosomal abnormalities range from 6 to 18%. Most of these chromosomal anomalies are restricted to the gonosomes, though there is concern that other, more subtle genetic diseases secondarily associated with male-factor subfertility or infertility will be transmitted.

As a result, genetic testing is becoming an integral part of patient assessment and treatment planning. One common test conducted prior to ICSI is for cystic fibrosis carrier status for patients presenting with congenital bilateral absence of the vas deferens (CBAVD), which affects 2% of infertility patients and 6–10% of all obstructive azoospermia patients. Studies indicate that approximately 65—70% of such patients are either mildly affected with or are carriers of cystic fibrosis, an autosomal recessive disease associated with respiratory system disease and exocrine pancreatic insufficiency.

Practitioners generally want to give these patients as much genetic information as possible. Clinical researchers hope to provide more-accurate genetic counseling for the infertile male. However, as gene function data become rapidly available it is sometimes difficult for physicians to distinguish between clinical research and clinical diagnostic applications of specific testing protocols. It is a challenge for the field to identify what information is useful in a research setting versus that which is appropriate to share with patients.

Clinical research is unraveling the genetic basis of male-factor infertility. For example, studies have led to the association of sperm and testicular mitochondrial deletions with severe male-factor infertility in up to 36% of the individuals studied. A similar correlation pertains to repeat expansion in exon 1 of the androgen receptor gene and male-factor infertility in up to 18% of the azoospermic or oligozoospermic men studied. One area of increased focus is understanding the significance of Y-chromosome deletions in the context of male-factor infertility.

Y-Chromosome Microdeletions

Cytogenetic and deletion analysis using polymerase chain reaction (PCR) amplification of sequence tagged sites (STSs) in DNA from azoospermic and oligozoospermic patients have led to the identification of at least four azoospermia factor (AZF) regions on the Y cromosome, namely, AZFa, AZFb, AZFc, and AZFd.

Beginning with the cloning of functional copies of the RBM and DAZ gene families from AZFb and AZFc, respectively, researchers have described numerous Y-linked genes whose expression is restricted to the testes. To date, more than 6000 patients have been screened employing from 1 to 131 STSs. The screening has resulted in microdeletion detection rates ranging from 3% to almost 30%.

One critical factor in developing a clinical diagnostic or clinical research screening protocol for Y-chromosome microdeletions is to screen a control, fertile population in order to identify polymorphic loci and loci that amplify non-Y-linked regions. STSs found to be deleted in this fertile control population should not be included in a screening protocol. The microdeletion detection rate does not necessarily improve with screening of large numbers of STSs. It is better to select STSs based upon scrutiny of the literature and then in-house validation. Secondly, it is a mistake to screen only infertile males with azoospermia and severe oligozoospermia. Men presenting with moderate to mild oligozoospermia may also have microdeletions in regions AZFb, AZFc, or AZFd.

Clinical research indicates that pathology-associated microdeletions occur at approximately similar rates in regions AZFb, AZFc, and AZFd. Although less than 10% of all pathology-associated microdeletions occur in region AZFa, 75% of the patients presenting with deletions in this region have severe congenital oligospermia, and 25% have partial spermatogenic arrest. Large microdeletions involving multiple AZF regions result in equally severe phenotypes. Patients with AZFb and AZFc microdeletions present with azoospermia, or oligozoospermia. Patients with AZFd microdeletions may have sperm counts ranging from azoospermia to normal sperm counts with abnormal sperm morphology.

A growing number of laboratories are screening for pathology-associated Y-linked STSs. An ideal study should cover all pathology-associated regions with thorough controls and careful data analysis followed by genetic counseling. In the first genetic screening of a population of ICSI-derived male children and their fathers, it was shown that microdeletions are heritable.^1,2 Males carrying only microdeleted Y cromosomes will transmit the identical mutation or an enlarged mutation to any sons conceived by ART, whereas males who are germ-line mosaics may produce sons who carry the microdeletion or sons with intact Y cromosomes. In this scenario, couples who produce multiple embryos by ART have the option of preimplantation genetic diagnosis.

Detection System

One outcome of these studies was the collection of sufficient data to configure the Y-Chromosome Deletion Detection system, developed by Promega Corp. (Madison, WI). The system employs multiplexed PCR and includes 18 primer pairs for nonpolymorphic STSs, grouped in four multiplex master mixes (see Table I). Figure 1 is a map of the Y cromosome showing the position of the STS primers.

Figure 1. A map of the Y cromosome showing the position of the STS primers in Promega's detection system.

One of the concerns with any multiplexed PCR system is random amplification failure. The primers of a multiplex system must have similar melting temperatures and should be designed to avoid secondary structure. It is vital to avoid nonspecific primer interaction, especially considering that each primer pair added increases the probability of primer-dimer formation. It is also challenging to find multiple primer pairs that amplify reliably with the same magnesium and nucleotide concentrations.

Figure 2. Multiplexed analysis of wild type male and female genomic DNA markers. Lanes 1–4 show the amplification products from reactions using multiplex master mix A with male DNA (lanes 1 and 2), female DNA (lane 3), and no DNA (lane 4). Lanes 5–8 show the amplification products from reactions using multiplex master mix B with male DNA (lanes 5 and 6), female DNA (lane 7), and no DNA (lane 8). Lanes 9–12 show the amplification products from reactions using multiplex master mix C with male DNA (lanes 9 and 10), female DNA (lane 11), and no DNA (lane 12). Lanes 13–16 show the amplification products from reactions using multiplex master mix D with male DNA (lanes 13 and 14), female DNA (lane 15), and no DNA (lane 16). Markers (M) are the 50 base pair DNA step ladder. The gel is 4% NuSieve 3:1 agarose.

During development of the detection system, a primary focus was optimizing the selection of primers to ensure that the multiplex mixes would perform reliably. In subsequent testing, the system has been shown to be robust and perform reproducibly. Figures 2 and 3 show examples of the banding profiles produced by using the multiplexes on a control male genomic DNA, on female DNA, and on DNA samples showing deletions in the Y cromosome. Analysis of the PCR product is by agarose gel electrophoresis. Deletions in the Y cromosome are detected by the absence of one or more amplification bands.

Figure 3. Y-chromosome deletion analysis, showing the amplification products from reactions using multiplex A (lanes 1 and 2), multiplex B (lanes 4 and 5), multiplex C (lanes 7 and 8), and multiplex D (lanes 10 and 11). Control human male genomic DNA from Promega (lanes 1, 4, 7, 10) is compared with a test male genomic DNA (lanes 2, 5, 8, 11). Markers (M) are the 50 base pair DNA step ladder. Nothing was loaded in lanes 3, 6, or 9. The gel is 4% NuSieve 3:1 agarose.

In any multiplexed PCR system there is a concern that random dropout of one amplification band or preferential amplification of smaller PCR products will invalidate the results. To reduce this concern, primers used in the detection system for adjacent STS regions are divided over the four multiplex mixes. Research has indicated that many of the deletions found in the Y cromosome will span multiple STSs. If a PCR product for a specific STS is absent in one reaction, frequently adjacent STSs in the other multiplexes will also be absent. Such a result indicates a real deletion in the Y cromosome. It would be very unlikely to find multiple noncontiguous deletions.

Each of the system's multiplex mixes includes an internal X-specific amplification control. This band will amplify even if the entire Y cromosome is absent, but will not amplify without the addition of template DNA.

Thermocycling protocols have been developed for the two most common designs of thermocycler—oil and no-oil (heated lid) systems. One thermocycling profile works for all the multiplex mixes, enabling the reactions to be performed at one time.

References

1. M Kent-First et al., "The Incidence and Possible Relevance of Y-Linked Microdeletions in Babies Born after Intracytoplasmic Sperm Injection and Their Fathers," Molecular Human Reproduction 2, no. 12 (1996): 943–950.

2. MG Kent-First et al., "Infertility in Intracytoplasmic-Sperm-Injection-Derived Sons," The Lancet 348, no. 9023 (1997): 332.

Marijo Kent-First, PhD, is an adjunct professor of obstetrics and gynecology at the University of Wisconsin (Madison), and new technology leader at Promega Corp. (Madison, WI). Angela Ryan, MS, is senior product manager for molecular diagnostics, Richard Schifreen is the business unit leader for molecular diagnostics, and Susan Frackman, PhD, is R&D project leader at Promega.

Back to the IVDT Nov/Dec 99 table of contents