1 The Fertility Centre, Private Bag 4711, Christchurch Women's Hospital, Christchurch, New Zealand and 2 Reproductive Medicine Unit, Department of Obstetrics and Gynaecology, University of Adelaide, The Queen Elizabeth Hospital, Woodville, Australia
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Abstract |
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Key words: congenital bilateral absence of the vas deferens/cystic fibrosis/genetic counselling/male infertility/obstructive azoospermia
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Introduction |
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Materials and methods |
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Analysis of CF gene mutations prior to treatment
Both partners provided a blood specimen, from which the leucocytes were separated and analysed, for a series of CF gene mutations (F508,
I506/7, G551D, G542X, R117H, R117C, R553X, and W1282X). The analysis was performed by Biochemical Genetics, Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, South Australia, based on the methods originally described to identify the CF gene (Kerem et al., 1989
). This group of mutations would be expected to identify about 80% of those mutations expected in the CF population. The identification of the poly T variant associated with cystic fibrosis mutations and discussion of the significance for potential parents required further analysis of the stored specimens for this variant from 1995 onward (Chillon et al., 1995
). Once the analyses were complete, clinical genetic counselling was provided.
Surgical sperm aspiration
The male partner was usually admitted on the day of oocyte retrieval. During the time course of this series, two surgical methods were employed for retrieval of spermatozoa. (i) Microsurgical epididymal sperm aspiration (MESA) under general anaesthesia with the use of an operating microscope was employed for the majority of cases (Oates et al., 1992). On one occasion, because no spermatozoa were harvested from either epididymis an open biopsy of the testicle was taken. (ii) Satisfactory reports of percutaneous epididymal sperm aspiration (PESA) (Shrivastav et al., 1994
) led to the introduction of PESA in 1996. PESA was offered as an alternative for those men preferring a less invasive procedure. A 21-gauge scalp vein needle was used to aspirate fluid directly from the epididymis under general or local anaesthesia. The patient recovery after this procedure involved bed rest for only 30 min if performed under local anaesthesia or 4 h if performed under general anaesthesia.
Preparation of spermatozoa
The samples were diluted with human tubal fluid medium (HTFM) containing 5% serum, centrifuged, and resuspended in HTFM before being incubated at 37°C in 5% CO2 until required. Whenever possible, supplementary spermatozoa were cryopreserved in several microstraws. A trial thaw of a representative straw was performed to assess if spermatozoa exhibiting some motility could be recovered. If satisfactory motile spermatozoa were recovered, the frozenthawed spermatozoa were used for subsequent treatment cycles. For a few couples, the sperm aspiration was performed electively prior to the treatment cycle or at the time of initial assessment and scrotal exploration, resulting in the use of cryopreserved spermatozoa in the initial IVF cycle.
Ovarian stimulation and embryology
The female partner underwent ovarian stimulation utilizing gonadotrophin-releasing hormone agonist (Lucrin®; Abbott Australasia, Kurnell, NSW, Australia) and human menopausal gonadotrophin (Pergonal® or Metrodin®; Serono, Frenchs Forest, NSW, Australia; Humegon®; Organon, Lane Cove, NSW, Australia) for ovarian stimulation. Ovarian response was monitored with serum oestradiol and transvaginal sonography, with transvaginal oocyte retrieval performed under ultrasonographic guidance. These protocols have been previously documented (Sathanandan et al., 1989). The procedure of ICSI has also been previously described and does not differ from that used for ejaculated spermatozoa (Payne, 1995
).
Embryos were transferred to the uterus on the second or third day after oocyte retrieval at the 2- to 8-cell stage. Supplementary embryos were considered for cryopreservation according to the usual laboratory criteria. Luteal support with intramuscular human chorionic gonadotrophin (HCG; Pregnyl®; Organon or Profasi®; Serono) injections following oocyte recovery. A serum pregnancy test was performed 16 days after the oocyte retrieval. Only clinical pregnancies defined by sonographic visualization of the gestational sac are included in this review. All pregnancies were followed up with the attending obstetrician providing a report of obstetric outcome and neonatal outcome.
Statistics
The results have been analysed using GraphPad InStat software, Version 2.04a, 1993, GraphPad Software with calculation of 95% confidence intervals and Fisher's exact test P values where appropriate.
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Results |
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Two pregnancies resulted from cycles in which pre-implantation genetic diagnosis (PGD) by blastomere biopsy was performed to identify the cystic fibrosis mutation F508 prior to embryo transfer. One couple were
F508 heterozygotes and the other included a female partner who was identified as a compound heterozygote (see below). A first trimester spontaneous abortion and a live birth resulted. Analysis of the products of conception and blood from the neonate confirmed the genotype expected from the pre-implantation diagnostic procedure (Cui et al., 1995
).
At the request of the parents the CF genotypes of two children derived from fathers who were F508 heterozygotes were tested and found to be heterozygotes. Both children are healthy, consistent with their heterozygous genotype.
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Discussion |
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The Wolffian duct abnormalities evident in CF and CBAVD indicate a common genetic link confirmed with the identification of CF gene mutations in both conditions. A review of 420 published cases of CBAVD indicated 19% had two mutations, 47% carried a single mutation, and in 34% no mutation could be identified (Lissens et al., 1996). Our experience supports these findings with a similar proportion of heterozygotes (60%), compound heterozygotes (20%) and 20% without a mutation. There is an increased prevalence of the R117H mutation within the CBAVD population. In this series, of the men with CBAVD (excluding the two with CF), six of the 25 (24%) of the CF chromosomes identified were R117H. This compares with less than 0.3% incidence of R117H in those patients with classic CF and is similar to the 22% initially reported in CBAVD (Gervais et al., 1993
).
Men with CBAVD but without CF gene mutations have a high incidence of urinary tract malformations (Dork et al., 1997). This group with urinary tract anomalies represent a separate clinical entity not related to CF and with a different embryological pathogenesis. In our study the five men without a CF mutation underwent ultrasound assessment of their renal tract and two of them had unilateral kidney agenesis.
The protein encoded by the CF gene is designated CF transmembrane conductance regulator (CFTR). In normal individuals, five, seven or nine thymidine (T) nucleotides can be identified at the intron 8exon 9 acceptor splice site of the CFTR gene (Chillon et al., 1995). The efficiency of splicing of exon 9 is thought to modify the phenotypic expression of the CF genotype. Individuals homozygous for the 5T allele have CFTR mRNA resulting in the production of non-functional CFTR protein. Some mutations are usually observed with the same T variant;
F508 is exclusively associated with 9T as was observed in this series. R117H occurs with two variants 5T and 7T resulting in different phenotypes although no 5T variant of R117H was identified in this series.
Genetic counselling
Genetic counselling prior to conception provides an estimate of the likelihood of inheriting a CF mutation from each parent. The probable phenotype of the child is calculated based upon the female partner's genotype, the severity of the mutation identified in the male partner, and the presence of intron 8 splice site variant. Even if CF mutation screening is negative there remains a small possibility that the individual carries an undetected mutation. For couples in whom the risk of CF is unacceptably high, prenatal diagnosis or pre-implantation genetic diagnosis can be offered. Pre-implantation genetic diagnosis has been successfully used for F508 and W1282X but not for the less common mutations.
Female heterozygotes carry a single CF gene mutation and have no signs of cystic fibrosis. No female equivalent of CBAVD exists due to the different embryological origin of the definitive female reproductive tract. One female compound heterozygote was identified. Her genotype (G551D/R117H) comprised a severe CF mutation (G551D) combined with R117H and the 7T allele identified on both chromosomes. She reported no past history of CF symptoms, and the assumption could be made that the R117H was present on a chromosome with an efficient intron 8exon 9 acceptor splice site. As her partner was a F508 heterozygote, pre-implantation genetic diagnosis by blastomere biopsy with PCR amplification was performed to avoid the transfer of a
F508 compound heterozygote embryo. Fifty per cent of such embryos would be
F508/G551D and likely to result in a child with CF. The remaining
F508 compound heterozygote embryos would be
F508/R117H compound heterozygotes that would be expected to confer a mild phenotype to the child. Transfer of embryos without the
F508 mutation would confer a G551D or R117H heterozygote genotype. The presence of an unidentified mutation together with
F508 in the male partner was a remote possibility which could also result in a compound heterozygote. Despite establishment of a pregnancy, spontaneous miscarriage occurred. Analysis of the products of conception confirmed the expected genotype with absence of
F508. The second couple for whom pre-implantation diagnosis was performed were
F508 heterozygotes. Transfer of homozygous normal embryos resulted in a livebirth of a girl with the genotype confirmed from cord blood.
ICSI outcomes
Earlier studies indicating poor oocyte fertilization rate and embryo implantation rate are in contrast to this series. Our results are comparable to those with ejaculated spermatozoa, and the implantation rates are higher than those achieved with routine IVF in our clinic (Payne et al., 1994).
For the two couples in whom the male partner were carriers of either the R117H or R117C CF mutation, pregnancy did not occur despite the transfer of 25 embryos over nine cycles. Embryos derived from males with compound heterozygote F508/R117H genotypes demonstrated a satisfactory embryo implantation rate. Larger studies will be necessary to establish if there is an association between outcome and specific CF mutations.
No significant differences were observed between the use of fresh and cryopreserved spermatozoa, however previously reported smaller series have noted a possible adverse effect of cryopreservation (Devroey et al., 1995; Nagy et al., 1995
). Our own concurrent larger series of treatment cycles including other causes of obstructive azoospermia has not demonstrated a significant reduction in oocyte fertilization rate and embryo implantation rate. Couples are now advised to consider cryopreservation of spermatozoa prior to treatment provided that the post thaw analysis has confirmed motile sperm recovery.
Only one of the 17 frozen thawed embryos transferred has resulted in a live birth. However, substantially larger numbers of embryos will need to be transferred before analysis of outcomes will be possible.
The clinical examination and follow-up of children born to couples with CBAVD or CF is essential to understand the variable phenotypic expression of CF gene mutations. For most couples in whom the male has CBAVD, the male is a carrier of a severe CF gene mutation and the female tests negative for the group of mutations screened. Therefore the offspring are expected to be asymptomatic (homozygous negative or asymptomatic heterozygotes). No significant congenital anomalies have been identified. The eight boys born to CBAVD fathers were examined and found to have a palpable vas deferens as expected.
For the two couples in whom the male had been diagnosed with cystic fibrosis, three oocyte retrieval cycles were completed. Sperm recovery, sperm cryopreservation, and the ICSI treatment cycle results were all comparable to the CBAVD men without clinical cystic fibrosis. These couples both conceived; however, one resulted in a miscarriage late in the first trimester, the other pregnancy resulted in the birth of a healthy infant. Historically a few reports of fertile CF males have been published. The demonstration of satisfactory spermatogenesis and this successful delivery of a healthy infant adds to the evidence confirming the ability of males with CF to father children.
It is concluded that the advice and treatment options available for infertile couples in whom men have a diagnosis of CBAVD have changed substantially in the course of this series. Analysis of both partners' medical and family history, cystic fibrosis genotype, and genetic counselling is required before initiating treatment. The presence of cystic fibrosis mutations is an important part of the assessment to permit adequate genetic counselling and the use of pre-implantation genetic diagnosis to reduce the likelihood of a child with cystic fibrosis may need to be considered. Surgical sperm aspiration and the use of fresh or frozen spermatozoa provide outcomes similar to those achieved with ejaculated spermatozoa. Cystic fibrosis mutations in the male partner do not appear to compromise oocyte fertilization, embryo implantation rates, or the opportunity for healthy live births. However the total number of live births reported remains small and continued study will be required to provide sufficient information to counsel couples contemplating treatment.
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Acknowledgments |
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Notes |
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References |
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Submitted on February 25, 1999; accepted on November 4, 1999.