119
Vol. 4, No. 2
REVIEW PAPER
Cystic fibrosis as a cause
of infertility
Katarzyna Jarząbek2, Monika Zbucka2, Witold Pepiński3, Jacek
Szamatowicz4, Jan Domitrz2, Jerzy Janica3, Sławomir Wołczyński1,2,
Marian Szamatowicz4
2
Department of Gynecological Endocrinology,
3
Department of Forensic Medicine,
4
Department of Gynecology,
Medical University of Białystok, Poland
Received: 15 November 2003; accepted: 16 March 2004
SUMMARY
Cystic fibrosis (CF) is one of the autosomal recessive diseases, caused
by mutations in a gene known as cystic fibrosis transmembrane regulator
(CFTR). The majority of adult males with CF (99%) is characterized by
congenital bilateral absence of vas deferens (CBAVD). CBAVD is encountered in 1-2% of infertile males without CF. Females with CF are found
to be less fertile than normal healthy women. In females with CF, delayed
puberty and amenorrhoea are common due to malnutrition. CFTR mutations are also associated with congenital absence of the uterus and vagina
(CAUV). The National Institutes of Health recommend genetic counseling
for any couple seeking assisted reproductive techniques with a CF male or
obstructive azoospermia which is positive for a CF mutation. Reproductive
Biology 2004 4 (2): 119-129.
Corresponding author: Department of Gynecological Endocrinology, Medical University of
Białystok, ul. M.C. Sklodowskiej 24A, 15-276 Białystok, Poland, e-mail: [email protected]
1
Copyright © 2004 by the Society for Biology of Reproduction
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Cystic fibrosis and infertility
Key words: cystic fibrosis (CF), cystic fibrosis transmembrane regulator
(CFTR), congenital bilateral absence of vas deferens (CBAVD), congenital
absence of the uterus and vagina (CAUV).
INTRODUCTION
A qualitative diagnosis of infertility requires attention to female and male
physical abnormalities, endocrine anomalies (their hormonal and genetic
background) and genetic conditions of both partners that interfere with
reproduction. Many genes are likely to be involved in the complex process
of reproduction [20]. Genetic and non-genetic causes of infertility are increasingly being identified.
The main genetic causes of male infertility diagnosed are microdeletions
of the Y chromosome (AZF region) connected with oligoasthenospermia or
aspermia as well as mutation of cystic fibrosis transmembrane conductance
regulator (CFTR) gene which is connected with aspermia and congenital
bilateral absence of vas deferens (CBAVD; [7]).
The cystic fibrosis gene encodes a cell membrane protein
The CFTR gene, located at chromosome 7, spans about 250 kb of genomic
DNA and contains 27 exons [34]. In 1989 it was the first anion channel to be
identified by positional cloning and sequencing [24]. The product of CFTR
gene regulates airway chloride transport. It is also a regulator of sodium
and water transport across the epithelium and perhaps acts as a transporter
of a still undefined substrate, or substrates [26, 30].
CFTR gene is a unique member of a large ATP binding cassette (ABC)
family of proteins known either as “ABC transporters” or “Traffic ATPases”
[8, 10]. CFTR protein is a single polypeptide chain of 1480 amino acids that
supposedly folds into five domains: two transmembrane-spanning domains
(TMS1 and TMS2) with tandem repetition of six putative transmembrane
helices also called membrane-spanning domains (MSD1 and MSD2), two
nucleotide-binding domains (NBD1 and NBD2), linked by regulatory domain (R) containing numerous phosphorylation sites (fig.1; [24]).
Jarząbek et al.
121
Fig.1. The structure of cystic fibrosis transmembrane regulator (CFTR). This
membrane protein is composed of membrane-spanning domains (MSD1 and
MSD2), nucleotide-binding domains (NBD1 and NBD2) and regulatory domain
(R) containing numerous phosphorylation sites.
Activation of CFTR is a multistep process that requires calcium and
phpspholipid-dependent protein kinase C (PKC) and cAMP-dependent
protein kinase A (PKA) and a high ATP/ADP ratio. Phosphorylations by
kinases and ATP hydrolysis or binding are crucial steps for activating the
channel [12]. Phosphorylation of CFTR protein occurs only in cytoplasmic R domain, whereas ATP binding or hydrolysis occurs in one of either
NBD. ATP was found to be hydrolyzed during gating activity of CFTR,
but a mechanistic link between ATP hydrolysis and specific step in CFTR
gating is still unexplained.
Cheung et al. have reported an interaction between CFTR and aquaporin-9
(AQP-9). The CFTR and AQP-9 were found in the luminal membrane of the
epididymis, where they play a crucial role in the formation of the epididymal
fluid and controlling water permeability. AQP-9 alone causes an increase of water permeability in the epididymis, which is further potentiated by CFTR [4].
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Cystic fibrosis and infertility
CFTR mutations
Over 1000 mutations reflecting all classes of genetic mutations have been
identified in CFTR gene and these mutations are located in all domains of
the protein. The result of mutations of the CFTR gene is multi- organ disease
- cystic fibrosis (CF). CF is the most common autosomal recessive disorder
in which abnormal regulation of epithelial Cl- channels is associated with the
pathophysiology of the disease. It is apparent that CFTR dysfunction results in
a range of effects which may contribute to clinical phenotype. The hallmarks of
CF include thick and dehydrated airway mucus, pancreatic insufficiency, bile
duct obstruction, infertility in males, reduced fertility in females, high sweat
Cl-, intestinal obstruction, nasal polyp formation, and chronic sinusitis [6].
CFTR mutations are divided into 5 classes. Class I mutations (null mutations) do not produce CFTR protein because of premature stop signal in
the CFTR DNA. Class II mutations lead to production of a CFTR protein,
but this protein attains an unstable structure shortly after translation in the
endoplasmic reticulum. Class III is based on production of a CFTR with
reduced chloride transport because of abnormal regulation of the chloride
channel. Class IV mutations partially reduce chloride conductance through
CFTRs. Finally, class V mutations lead to a severe reduction in normal
CFTR protein form [25].
The most common mutation associated with CFTR (responsible for 70%
of abnormal CF gene) is a three base-paired deletion that produces an inframe deletion of a phenyloalanine (F) at position 508 (NBD1 domain). Its
frequency varies between ethnic groups (82% patients with CF in Denmark,
32% in Turkey; [22]). This mutant protein of CFTR is known as Δ F508 CFTR.
The absence of phenyloalanine at position 508 appears to be associated with
abnormal folding of CFTR protein during translation, an abnormality recognized by cellular chaperone proteins that mediate intracellular degradation by
proteosomal and other pathways prior to transit of mutant protein to plasma
membranes [3, 19, 32]. This mutation leads to a drastically reduced level of
CFTR protein expressed on the plasma membrane of exocrine epithelia. If
CFTR Δ F508 protein reaches the cell surface without degradation, it exhibits
a partial function as a cAMP-activated chloride channel.
Jarząbek et al.
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Males with CF
Majority of adult males with CF (99%) has CBAVD. CBAVD is also
encountered in 1-2% of infertile males without cystic fibrosis [2]. Men
with CBAVD but without CF gene mutations have a high incidence of
urinary tract malformations [9]. The group with urinary tract anomalies
represents a separate clinical entity not related to CF and with different
embryological pathogenesis. Some forms of infertility found in otherwise healthy men have also been reported to be associated with CFTR
mutations, especially obstructive azoospermic conditions such as congenital bilateral absence of vas deferens (CBAVD), unilateral absence
of vas deferens, epididymal obstruction and bilateral ejaculatory duct
obstruction with concomitant seminal vesicle anomalies [33]. CBAVD
is caused by a disruption in the vas deferens, a Wolffian duct derivative. Among 420 cases of men with CBAVD, 19% were carriers of two
alleles mutations of CFTR, 47% of a single allele mutation and 34% of
no identified mutation [18].
The diagnosis of CBAVD is based on the presence of azoospermia in
subjects with normal or small size testes, non-palpable vas deferens, the
characteristic ultrasonography view and changes in the physical and biochemical properties of ejaculate (small volume, low pH, and low fructose
concentration; [17]). Genital abnormalities may occur early in CF, but are
less common diagnosed than in adults. They are found more often in pancreatic insufficient than in pancreatic sufficient CF patients [2].
Mutations in the cystic fibrosis transmembrane conductance regulator
gene are a relatively frequent cause of male infertility. Depending on their
molecular consequences, CFTR mutations may either result in a typical cystic fibrosis, or in an atypical (often monosymptomatic) forms of CF such as
congenital absence of the vas deferens (bi- or unilateral), bilateral ejaculatory
duct obstruction or bilateral obstructions within the epididymides. Infertile
men might have an elevated risk of being carriers of a mutation within the
CFTR gene, in comparison with the general population. Therefore, it is
recommended to conduct CFTR mutation tests in males with idiopathic
infertility prior to reproduction techniques.
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Cystic fibrosis and infertility
CFTR screening includes the most frequent CFTR mutations, for
example in the German population: ΔF508, R347P, G542X, S549I, N, R
(A→C), G551D, R553X, N1303K, 3849+10kbC→T [11]. In the French
population high frequencies of the ΔF508 mutation (44.7%), the T5 allele
(36.2%) and R117H mutation (19.1%) were observed [13]. The data above
can be different for other ethnic groups.
Additionally, Chu et al. have identified the mutations within the polypyrimidine tract of the CFTR intron 8-splice acceptor site (IVS8-T tract),
consisting of five, seven, nine thymidines (5T, 7T, 9T). The 5T variant
produces a lower level of normal CFTR mRNA transcripts than the 7T and
9T alleles and is associated with disseminated bronchiectasis, CBAVD and
epididymal obstruction [5]. Arduino et al. have described a CBAVD patient
with a compound heterozygosity in the CFTR gene for a stop mutation
W1282X and a new missense mutation P499A. The P499A was found as a
mild mutation, limited to the development of Wolffian duct derivatives and
was revealed only in combination with a severe CFTR mutation [1].
In our investigations a sample patient is screened for the presence of 31
common CF mutations, including the 24 most common CF mutations worldwide, as identified by the CF consortium. This mutation detection system
employs a rapid, single tube, multiplex DNA technology associated with
CF incorporating polymerase chain reaction (PCR), oligonucleotide ligation
assay (OLA) and sequence-coded separation (SCS) using the fluorescent
technology. Products from the multiplex OLA are detected in a capillary
electrophoresis, 4color fluorescent ABI 310 DNA Analyzer (Applera, USA).
Sequence-Coded Separation identifies each ligation product as a unique
combination of electrophoretic mobility and color.
Testicular biopsies of post-pubertal men with CF have shown abnormal histological findings, such as pathological spermatogenesis and increased number
of dysmorphic spermatozoa, yet testicular histology specimens obtained from
men with isolated CBAVD have shown completely normal spermatogenesis.
This fact could be a result of nutritional deficiency, gonadotoxic drugs (e.g.
antibiotics, corticosteroids) or systemic illnesses rather than a primary defect
in spermatogenesis. There is no evidence of association between CFTR gene
mutations and the non-obstructive azoospermia phenotype [21].
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Fig. 2. Screenig of cystic fibrosis (CF mutation). Detection of mutation ΔF508
(peak within dotted ellipse) using CF Multiplex DNA Assay (Applera, USA).
The use of percutaneous epididymal or testicular sperm aspiration (PESA,
TESA) and intracytoplasmic sperm injection (ICSI) has allowed the attainment
of pregnancies in this group of patients [27]. If any CF mutation is detected in
a patient, CFTR screening should involve the female partner [11]. The female
partner of a man with CFTR-mediated anomalies of the genital tract may be
heterozygous purely by chance (4–5% in many countries).
Since infertility in many cases can be treated nowadays by assisted
reproductive technology, there is an increased risk of having children with
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Cystic fibrosis and infertility
CF if the male and female are carriers of CFTR mutations. Couples should
be informed about the risk and risk prevention including pre-implantation
diagnosis. Follow-up studies of children born to these couples are mandatory,
whether male infertility is linked to CF or not [27].
Females with CF
Females with CF are found to be less fertile than normal healthy women. In CF
females delayed puberty and amenorrhoea are common due to malnutrition as
the main cause [29]. Delayed pubertal increments of serum gonadotrophin and
sex steroids suggest late maturation of the reproductive endocrine system [23].
The patients who were homozygous for the most common mutation ∆F508
and those with pathological glucose tolerance test (OGTT) were significantly
delayed in menarcheal age. The majority of the patients had essential fatty
deficiency (EFAD) which may cause pubertal delay [15].
The low fertility in CF females is known to be caused mainly by tenacious impermeable cervical mucus, which does not undergo the typical
changes during menstrual cycle, due to defective CFTR protein expressed
in the cervix [16]. Some CF women do not ovulate. They have higher total
serum testosterone concentration and signs of insulin resistance similar to
those found in women with polycystic ovarian syndrome. Anovulation due
to malnutrition and catabolism has been suggested as a secondary cause
of infertility. CF patients might have inflammation in their tissues prior to
infection with an imbalance of proinflammatory cytokines versus the antiinflammatory ones.
Recently CFTR mRNA expression has been found in areas of rat hypothalamus which are involved in the regulation of sexual maturation and
reproduction. These areas contain high concentrations of GnRH and estrogen-rich neurons. CFTR might increase acidification of synaptic vesicles
and for that reason it plays an important role in central regulation of sexual
maturation and fertility [14]. Ovulation in these chronically ill women may
also be influenced by physiological and psychological stress. CFTR mutations are also associated with congenital absence of the uterus and vagina
(CAUV; [31]). Since the embryologic development of the Mullerian ducts
Jarząbek et al.
127
directly depends on the prior normal development of the Wolffian ducts, the
same gene products may be necessary for normal embryologic development
of both duct’s systems.
The incidence of the 33 CFTR mutations found in the patients with
CAUV (8%) was twice as high as that found in the general population (4%),
but much less than the incidence of CFTR mutations in men with CBAVD
(80%). This data suggests that it is unlikely for CFTR mutations to cause
CAUV in females as they cause CBAVD in males. Furthermore, the data
suggests that CAUV in females may be the same disorder as CBAVD in
males who do not have CFTR mutations.
National Institutes of Health recommends genetic counseling for any
couple attempting assisted reproductive techniques, when the man has CF
or obstructive azoospermia and is positive for a CF mutation [28]. Clinical
genetic conditions of a family considering assisted reproduction of infertility
can be established by evaluating the full family history, documenting pregnancy, fetal, neonatal and pediatric loss of life as well as by cytogenetic studies
of the couple and DNA mutation analysis for cystic fibrosis mutations.
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