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If your patient is unable to get pregnant because her partner has cystic fibrosis or congenital bilateral absence of the vas deference, assisted reproductive technology offers a solution. But these disorders have genetic implications that you need to keep in mind when advising such couples.
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If your patient is unable to get pregnant because her partner has cystic fibrosis or congenital bilateral absence of the vas deferens, assisted reproductive technology offers a solution. But these disorders have genetic implications that you need to keep in mind when advising such couples.
Cystic fibrosis (CF) is a common disorder found in approximately one out of 1,600 people of Northern European descent.1 Almost all men with CF lack vasa deferentia bilaterally.2 Another disorder, congenital bilateral absence of the vas deferens (CBAVD), is found in approximately 1% of infertile men and in a higher percentage of those presenting with azoospermia.3
Until recently, men with CF or CBAVD had virtually no chance to become fathers, but with today's technology that's now possible.4 Clinicians involved in reproductive medicine need to understand the facts and appreciate the nuances of these two disorders in order to counsel couples suffering from infertility. In this article, I will discuss the genetic issues involved and how we can help women and their affected partners realize conception.
It was discovered in 1968 that the reason for infertility in adult males with clinically recognized CF was an absence of both vasa deferentia.5 Spermatogenesis is normal in such men, but vasal aplasia leads to obstructive azoospermia.
CF is the most common lethal autosomal recessive disease found in people of Northern European descent. Among asymptomatic individuals with this background, the carrier frequency is 4% to 5%. Rommens and coworkers found that the CF gene is located in a region on the long arm of chromosome 7, locus 31.6 A 250-kb fragment of this gene encodes a protein called cystic fibrosis transmembrane conductance regulator (CFTR). This is a membrane-bound molecule that assists in the transmembrane transport of chloride ions, to maintain an optimal state of hydration in epithelial-lined lumina. If CFTR is dysfunctional, secretions in pulmonary small airways and in the pancreatic ducts become tenacious and obstruct those structures, leading to respiratory and pancreatic complications.
Over 800 mutations have been identified in the CF allele, the most common of which is
F508. This mutation represents an in-frame three base pair deletion that completely removes a phenylalanine residue from CFTR, substantially limiting its functional capacity. Some other mutations, like R117H, have a much less significant impact on the integrity and ability of the resultant CFTR protein. It is the mildest mutation of the two that a patient inherits that determines the clinical nature of disease.
If two severe mutations coexist in an individual, as often happens when CF patients are homozygous for
F508, the disease will be extreme in nature, with both pulmonary and pancreatic dysfunction as well as vasal agenesis. But when the abnormalities of the CF gene are not as severe, the disease is less harsh. In these situations the patient may have adequate pancreatic function but compromised respiratory status and no vasa. In short, the phenotypic spectrum of CF depends upon the exact abnormalities in the two CF alleles and these determine the severity of disease.
The same concepts are useful for understanding congenital absence of the vas deferens.7 Men with CBAVD have normal pulmonary and pancreatic function; their only clinical similarity to CF is vasal agenesis. The first recognition of their condition may be when they present as the male partner in an infertile marriage. Although they are free of most symptoms of CF, 65% to 80% of men with CBAVD have at least one detectable mutation or abnormality in their CF alleles.8-10 This is obviously higher than the known carrier frequency in the general population and demonstrates that many cases of CBAVD are mild phenotypic expressions of CFTR dysfunction.
The CFTR pool, however, is not as depleted or dysfunctional in men with CBAVD as in those with CF-related pulmonary or pancreatic disease in addition to vasal agenesis. It appears that CBAVD and CF are at two opposite ends of the phenotypic range determined by CFTR aberration. One of the most common CF gene abnormalities found in men with CBAVD is the 5-thymidine variant of the polythymidine tract in the splice acceptor site of intron 8 that precedes exon 9 (IVS8-5T).11,12 This variant is found in approximately 5% of the general population and limits the pool of mature mRNA that includes exon 9 message.
Much of the transcribed exon 9 is spliced out during processing of the nascent mRNA and the amino acid derivatives of exon 9 are therefore not included in the final CFTR protein, leading to a major disruption of its functional capacity. If a 5T allele is coupled with a mutation in the opposite CF gene, the total pool of normal CFTR is significantly reduced to the level where vasal morphogenesis may be compromised. For example, in a series of men with CBAVD, a 5T allele was found in 24 of 64 men (38%) and this was associated with a recognized mutation on the opposite allele (13 of 64 men, 20%).13 In this same series, 72 mutations were detected when both direct sequencing and 5T analysis was performed. This frequency is significantly higher than that detected by a routine panel searching for the 31 most common mutations in the CF population.
The spectrum of mutations in men with CBAVD is somewhat different from that found in the CF population. The genetics laboratory should be aware of the clinical diagnosis so that the testing can be more focused and precise.
It is likely that a couple complaining of infertility due to CF or CBAVD will nonetheless attempt pregnancy. If the woman carries a mutation, the offspring may inherit two mutations and could be affected by either CF or CBAVD, depending upon the exact abnormalities present (Figure 1). For this and other reasons, a man with CF or CBAVD should have CF mutation analysis.3 Depending on the findings, preimplantation genetic screening can be performed on any embryos generated during assisted reproductive procedures, to prevent the transfer of those harboring mutations in both alleles.
A close collaboration with our colleagues in medical genetics is important for estimating the risks and advising couples in this situation. Even though it is impractical to have your patient and her partner tested for every mutation and polymorphism described to date, the a priori risk estimate for the couple can be reduced to the order of 1 out of 400 by looking for a selection of the most commonly occurring abnormalities in the CF gene. The child will still be a carrier, but by itself this is not harmful. After all, one of 20 people of Northern European descent is a carrier. Concerns about increasing the carrier pool are unwarranted, as the effect on the world's carrier population is barely measurable when these couples choose to have children.
The diagnosis of CF or CBAVD also means others in the man's family, most importantly his siblings, may have CF mutations as well. If we assume that a male with CF or CBAVD has two identifiable CF gene anomalies, we can predict that 25% of his brothers and sisters will inherit no mutations from their parents, 25% will inherit the two CF allele abnormalities, and 50% will inherit one mutation from either father or mother. This puts these brothers and sisters at risk of either having some expression of disease themselves or of potentially having a child with CF or CBAVD if their spouse is an asymptomatic carrier. Family screening can identify these at-risk individuals.
A small percentage of men with CBAVD have unilateral renal agenesis and no identifiable CF mutations. These men probably have a completely different genetic etiology that does not involve the CF genes. It may be one that could lead to unilateral or bilateral renal agenesis in conceived offspring. The couple should be informed of this possibility, even though the exact chances of occurrence are unknown at present.
In normal male anatomy, spermatozoa are released from the Sertoli cells and begin their journey in the lumen of the seminiferous tubules. These initial passageways coalesce in the mediastinum of the testis to form the rete testis. From here arise six to eight efferent ductules that travel outside the confines of the testis and co-mingle in the first portion of the epididymis, the caput. The efferent ducts eventually merge into a single tubule that is highly coiled and successively forms the corpus and cauda epididymis.
Men who lack vasa nevertheless have the caput epididymis, because this structure derives from a different embryologic precursor than the corpus, cauda, vas, and seminal vesicle. In approximately 50% of cases of CBAVD, remnants of either the corpus or cauda may be present, continuing from the epididymal tubule proper as it emerges from the caput.
In men with these conditions, semen analysis shows a low volume (<1 mL) and semen is acidic (pH <7.0) and azoospermic. The seminal vesicles are aplastic or atrophic and if there is a residual tissue remnant, it is typically dysfunctional.14
Silber and colleagues pioneered microsurgical sperm aspiration for men with CBAVD, in combination with in vitro fertilization. My group extended this strategy to those with clinical CF.15 However, the overall success rate was poor in those early days, due to the limited ability of the sperm to fertilize a harvested oocyte during standard IVF. In data published by the Sperm Microaspiration Retrieval Techniques Study Group, the fertilization rate was only 7% and the ultimate pregnancy rate was 5%.16
The solution, again due to Silber, was to apply the new technique of intracytoplasmic sperm injection (ICSI), where a single sperm is mechanically placed into the retrieved oocyte.17 ICSI was able to overcome the sperm's inability to penetrate the outer oocyte membranes and deliver the genetic package to the interior of the oocyte, so the rates of success climbed dramatically. The high fertilization rate proved that the nucleus of the spermatozoan surgically harvested from patients with CF or CBAVD was perfectly capable of performing its part in the fertilization process if it could only get into the ooplasm.
ICSI is such a powerful tool in cases like these that we began to use cryopreserved epididymal sperm with efficacy equal to that of freshly retrieved sperm.18 This allows greater flexibility, because sperm harvesting does not need to happen at the time of oocyte retrieval.19 In addition, a single surgical intervention can provide a quantity of sperm for cryopreservation in several vials, to serve as the source of sperm for multiple ICSI cycles if necessary. Pregnancy rates now approach those routinely seen during ICSIapproximately 35% to 45% per cycle, depending on the female partner's fertility.
Microsurgical epididymal sperm aspiration (MESA) is carried out under local anesthesia and a small amount of IV sedation. A small incision is made in the tunic of the epididymis and a single epididymal tubule is isolated. A tiny hole in the anterior presenting surface is created and the fluid that exudes is gently aspirated into a sperm-friendly medium. The sample is analyzed, processed sparingly, and then subdivided into 6 to 10 cryovials (Figure 2). The morbidity from this open approach is minimal.
In the future, we may simply extract testis tissue to serve as the reservoir of sperm, that is, if frozen and thawed testis sperm is shown to be successful and free of long-term harm. For now, though, most embryologists prefer to work with sperm-containing fluid rather than sperm-containing tissue.
In conclusion, men with CF or CBAVD cannot father children naturally, but can do so using a combination of sperm retrieval and ICSI. We understand the genetic basis of the disorders and must ensure that proper testing and counseling take place prior to any intervention, so the parents-to-be are aware of genetic risks to conceived offspring.
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