Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing.

BACKGROUND: The present study was undertaken to examine the usefulness of both vitrification and assisted hatching (AH) on blastocysts that originate from embryos showing different qualities during their cleavage stage. METHODS: A total of 281 blastocysts were vitrified (93 vitrification-warming cycles) in a mixture of ethylene glycol-dimethylsulphoxide-Ficoll and sucrose using the Hemi-Straw (HS) carrier system. After warming, AH using the partial dissection technique was performed in 36 cycles. RESULTS: After warming and culture for 24 h, a total of 168 blastocysts (60%) was suitable for embryo transfers and a total of 25 ongoing pregnancies (27%) was obtained. Forty-nine transfers of 96 no-AH blastocysts and 36 transfers of 72 AH blastocysts resulted in an implantation rate of 13 and 22% respectively (P < 0.05). The percentage of transfers with at least one hatching blastocyst was significantly higher after application of AH (69 versus 33%) (P < 0.001). In all, 73 and 38% of blastocysts showing respectively optimal and non-optimal embryo development during the early stage were available for transfer (P < 0.001). Consequently, implantation rates of 19 and 6% were obtained after transfers of blastocysts showing respectively optimal and poor embryo development. CONCLUSIONS: Artificial opening of the zona pellucida after warming of vitrified blastocysts significantly improved the rate of transfers with hatched blastocysts and the implantation and pregnancy rates. The percentage of blastocysts that survived the HS vitrification procedure and were available for embryo transfer is related to their previous developmental quality.

Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification.

BACKGROUND: In 1996, with the introduction of sequential media, we set up a programme of cryopreservation of supernumerary morulae (day 4) and blastocysts (day 5) using a vitrification procedure. Our results showed that the efficiency of the vitrification method was dependent on the stage of embryo development and was negatively correlated with the expansion of the blastocoele. We postulated that a large blastocoele might disturb cryopreservative potential due to ice crystal formation during the cooling step. We analysed therefore the effectiveness of reducing before vitrification the volume of the blastocoelic cavity. METHOD: Day 4 and day 5 embryos were vitrified in 40% ethylene glycol-18% Ficoll and 0.3 mol/l sucrose before plunging the straws directly into liquid nitrogen. Artificial shrinkage of the blastocyst was achieved after pushing a needle into the blastocoele cavity until it contracted. RESULTS: The survival rate post-thawing of day 4 and intact day 5 embryos was correlated with the volume of the blastocoele. In the control group only 20.3% blastocysts or expanded blastocysts survived as compared with 54.5 and 58.5% with morulae and early blastocyst respectively. After puncturing the blastocoelic cavity, an increase in the survival rate of up to 70.6% was noted. The pregnancy rates were improved after the artificial shrinkage procedure (20.5%) compared with the control intact blastocyst group (4.5%) (not significant). After reduction of the blastocoelic cavity, a significant increase in the implantation rate per vitrified blastocyst was observed (12.0 versus 1.4% P < 0.01). CONCLUSIONS: Our results showed that survival rates in cryopreserved expanded blastocysts could be improved by reducing the fluid content. This was presumably because mechanical damage caused by ice crystal formation was avoided. These observations should be considered when establishing a strategy and a protocol for cryopreservation of day 5 embryos.

Preimplantation genetic diagnosis for sickle-cell anemia and for beta-thalassemia.

We developed single-cell polymerase chain reaction (PCR) assays for preimplantation genetic diagnosis (PGD) in couples carrying mutations in the beta-globin gene. With PGD the genetic status of an embryo obtained after intracytoplasmic sperm injection (ICSI) is determined by PCR analysis in single blastomeres, allowing only healthy embryos to be transferred to the uterus. We carried out nine PGD cycles using fluorescent PCR for two couples in whom the partners carried sickle-cell trait. Both couples achieved pregnancies, one of which was spontaneously aborted. We have developed two beta-thalassemia PGD protocols: one for the analysis of the 25-26delAA and the IVS2+1G>A mutation, and the other for the simultaneous detection of the IVS1+6T>C and the IVS1+110G>A mutations. For the second protocol, both non-labelled PCR and later fluorescent PCR were used. Both protocols were applied in clinical cycles (two non-labelled PCR cycles and one fluorescent PCR cycle) for two couples. The patient with the fluorescent PCR-PGD cycle became pregnant. Overall, the three fluorescent PCR assays were accurate and reliable with amplification efficiencies of minimum 93% and allele dropout (ADO) rates between 0 and 12%.

Preimplantation genetic diagnosis for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common defect in fatty acid oxidation. The disease is inherited in an autosomal recessive fashion (carrier frequency around 1 in 70) and probably affects as many as 1 in 10000 new-borns. Affected children usually present within the two first years of life with recurrent episodes of hypoketotic hypoglycaemia and lethargy leading to death in approximately 25% of the cases. One mutation (c985A-->G) accounts for approximately 90% of the carrier chromosomes. We developed a preimplantation genetic diagnosis (PGD) strategy for MCAD for a couple who had already lost two affected children. When tested on heterozygous lymphoblasts, the amplification efficiency was 67 out of 71 (94%) and the allele drop-out rate was 0 out of 67. The patient became pregnant after one PGD cycle during which two embryos were replaced. The twin pregnancy was checked by chorionic villus sampling (CVS) and was shown to be unaffected. The twins have been born and are healthy.

Two pregnancies after preimplantation genetic diagnosis for osteogenesis imperfecta type I and type IV.

Osteogenesis imperfecta (OI) is an autosomal dominant genetic disorder characterized by the presence of brittle bones and decreased bone mass (osteopenia), as a result of mutations in the genes that encode the chains of type I collagen, the major protein of bone. The clinical features of the disease range from death in the perinatal period to normal life span with minimal increase in fractures. The present report describes two polymerase chain reaction (PCR)-based assays allowing preimplantation genetic diagnosis (PGD) on the one hand for OI type I, the mildest form, and on the other hand for OI type IV, which is intermediate in severity between OI type I and OI type III. In the couple referred for PGD for OI type I, the female partner carried a 1-bp deletion in exon 43 of the COL1A1 gene, resulting in a premature stop codon in exon 46. The synthesis of too little type I procollagen results from such a non-functional or COL1A1 null allele. In the other couple, referred for PGD for OI type IV, the male partner carried a G to A substitution in exon 19 of the COL1A2 gene, which results in an abnormal gene product due to an alphaGly247 (GGT) to Ser (AGT) substitution (G247S). Both mutations result in the loss of a specific restriction enzyme recognition site and can therefore be detected by PCR amplification followed by restriction fragment analysis. PCR amplification of genomic DNA of the parents-to-be with one of the two primers fluorescently labelled, followed by automated laser fluorescence (ALF) gel electrophoresis of the amplified and restricted fragments, allowed a distinction between the healthy and affected genotypes. PCR on single Epstein-Barr-virus (EBV)-transformed lymphoblasts resulted in acceptable amplification efficiencies (87% and 85% for OI type I and OI type IV respectively) and the allele drop-out (ADO) rate was assessed at 11.5% and 11.1% for OI type I and OI type IV respectively. With research blastomeres, 100% amplification rates were obtained and no contamination was observed in the blank controls, which validated the tests for clinical application. Embryos obtained after intracytoplasmic sperm injection (ICSI) were evaluated for the presence of the normal genotype of the non-affected parent. For OI type I, two frozen-thawed ICSI-PGD cycles and two fresh ICSI-PGD cycles were carried out for the same couple. The transfer of two unaffected embryos in the last cycle resulted in a twin pregnancy. A twin pregnancy was also achieved in one clinical ICSI-PGD cycle for OI type IV.

Clinical application of preimplantation genetic diagnosis for cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease characterized by obstruction and chronic infections of the respiratory tract and pancreatic insufficiency. The gene was cloned in 1989 and the most frequent mutation was shown to be the delta F508 mutation. During PGD, embryos obtained in vitro are checked for the presence or absence of the mutation, after which only embryos shown to be free of the mutation are returned to the mother. Up to 1999, 48 intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) cycles had been carried out for PGD for CF in 24 couples, and different diagnostic tests had been used to select non-affected embryos. Thirteen patients became pregnant and 12 healthy babies have been born.

Embryo implantation after biopsy of one or two cells from cleavage-stage embryos with a view to preimplantation genetic diagnosis.

Preimplantation genetic diagnosis (PGD) can be offered as an alternative to prenatal diagnosis (PND) to couples at risk of having a child with a genetic disease. The affected embryos are detected before implantation by fluorescent in situ hybridisation (FISH) for sexing (X-linked diseases) and chromosomal disorders (numerical and structural) or by polymerase chain reaction (PCR) for monogenic disorders (including some X-linked diseases). The accuracy and reliability of the diagnosis is increased by analysing two blastomeres of the embryo. However, the removal of two blastomeres might have an effect on the implantation capacity of the embryo. We have evaluated the implantation of embryos after the removal of one, two or three cells in 188 PGD cycles where a transfer was done. The patients were divided into five groups: a first group which received only embryos from which one cell had been removed, a second group which received only embryos from which two cells had been removed, a third group which received a mixture of embryos from which one and two cells had been taken, a fourth group where two and three cells had been removed, and a fifth group where three cells had been removed. The overall ongoing pregnancy rate per transfer was 26.1%, the overall implantation rate per transfer was 15.2% and the overall birth rate was 14.2%. Although pregnancy rates between the groups cannot be compared because the second group (two cells removed) contains more rapidly developing and therefore 'better quality' embryos, an ongoing pregnancy rate of 29.1% and an implantation rate of 18.6% per transferred embryo in this group is acceptable, and we therefore advise analysing two cells from a > or =7-cell stage embryo in order to render the diagnosis more accurate and reliable.

Cumulative delivery rates after intracytoplasmic sperm injection: 5 year follow-up of 498 patients.

The use of life-table analysis for infertility data has the advantages of clarity and ease of application. Success rates per cycle have been reported, but not cumulative delivery rates for intracytoplasmic sperm injection (ICSI). We selected retrospectively 498 Belgian patients <37 years old, who had their first ICSI cycle between July 1992 and December 1993. Follow-up was till the end of October 1997. Outcome measure was any delivery >25 weeks. These couples underwent 963 ICSI cycles using fresh ejaculated spermatozoa. The indications for ICSI were long-standing severe male infertility or fertilization failure after conventional in-vitro fertilization (IVF). Cumulative delivery rates were calculated by life-table analysis and compared according to age groups and sperm quality. There were 298 deliveries within a mean rate per cycle of 31%. The average number of cycles required for a delivery was 3.15 (CI 2.88; 3.43). Twenty-three (4.6%) spontaneous pregnancies occurred after the patients had finished therapy. There was no significant difference between the sperm quality groups but delivery rates decreased significantly with increasing female age. The real delivery rate after six cycles was 60%, while the expected cumulative delivery rate was 86%. This life-table analysis may provide a means by which to counsel couples on the likelihood of a delivery following ICSI.

Controlled comparison of conventional in-vitro fertilization and intracytoplasmic sperm injection in patients with asthenozoospermia.

A controlled comparison between conventional in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) has been carried out for patients with

Preimplantation genetic diagnosis and sperm analysis by fluorescence in-situ hybridization for the most common reciprocal translocation t(11;22).

In this study we describe the pre-clinical development and clinical application of preimplantation genetic diagnosis (PGD) by fluorescence in-situ hybridization (FISH) for two non-related carriers (one male and one female) of the most common balanced reciprocal translocation: t(11;22)(q25;q12). For the couple with the female carrier, enumeration of the sex chromosomes in the embryos was also indicated (husband: 47,XXY karyotype). Four-colour FISH analysis was performed on six blastomeres from three embryos. No embryo transfer was possible because all the embryos were unbalanced. Three PGD cycles, with two-colour FISH, were carried out for the couple with the male translocation carrier. A total of 35 embryos were biopsied and diagnosed by FISH; nine out of the 35 embryos (25. 7%) were normal and seven of them were transferred (two embryos from the first and four from the third cycle), six out of 35 embryos (17%) were unbalanced, three out of 35 embryos (5.7%) were triploid or polyploid, 10 out of 35 embryos (28.6%) were mosaic and seven out of 35 embryos (20%) were chaotic. Diagnosis failed in 2.9% of the embryos. The spermatozoa of the male carrier were also analysed using three-colour FISH. Only 29.1% of the sperm cells seemed to be balanced or normal. By choosing probes lying on both sides of the breakpoints and by using a combination of sub-telomeric or locus-specific probes and centromeric probes, the use of three-colour FISH enabled detection of all the imbalances in sperm and/or cleavage-stage embryos in the patients. This may improve risk assessment and genetic counselling in the future for translocation carriers.

Fluorescent PCR and automated fragment analysis in preimplantation genetic diagnosis for 21-hydroxylase deficiency in congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease which is most often caused by a deficiency in steroid 21-hydroxylase. The disease is characterized by a range of impaired adrenal cortisol and aldosterone synthesis combined with an increased androgen synthesis. These metabolic abnormalities lead to an inability to conserve sodium and virilization of females. The most common mutation causing the severe form of CAH is a conversion of an A or C at nucleotide (nt) 656 to a G in the second intron of the steroid 21-hydroxylase gene (CYP21) causing aberrant splicing of mRNA. A couple was referred to our centre for preimplantation genetic diagnosis (PGD) for 21-hydroxylase deficiency in CAH. A PGD was set up to detect the nt656 A/C-->G mutation using fluorescent polymerase chain reaction (PCR) and subsequent restriction enzyme digestion and fragment analysis on an automated sequencer. Using DNA or single cells from the father, the normal allele could not be amplified. Non-amplification of the normal allele has been previously described in asymptomatic carriers, therefore the PCR was further developed using heterozygous lymphoblasts from the mother. The PCR was shown to be highly efficient (96% amplification), accurate (0% contamination) and reliable (0% allelic drop-out). The couple started PGD treatment and the second PGD cycle resulted in a twin pregnancy. The genotype of the fetuses was determined in our laboratory using chorionic villus sampling material using the method described here. Both fetuses were shown to be heterozygous carriers of the mutation, and two healthy girls were born.

Clinical experience of sex determination by fluorescent in-situ hybridization for preimplantation genetic diagnosis.

In our centre we started using fluorescent in-situ hybridization (FISH) technique for sexing in couples with sex-linked diseases in May 1995. Probes specific for chromosomes X, Y and 18 were applied, allowing us to detect simultaneously both gender and ploidy status. The efficiency of the FISH procedure is 90.4% per biopsied blastomere or 95.2% per biopsied blastomere with a distinct nucleus visible at spreading. Up to December 1997, we treated 15 couples (20 treatment cycles) at risk for X-linked recessive disease and two couples with Yq deletion (two treatment cycles) with the aim of transferring only female embryos. In one cycle, no embryos suitable for biopsy were obtained and in five cycles no normal female embryos were available at diagnosis. In the remaining 16 cycles, transfer was possible and six pregnancies ensued: one miscarriage has occurred and six children have been born from the other five pregnancies. The implantation rate (fetal sacs) per transferred embryo was 20.8%. In 98 (61%) of the 161 diagnosed embryos, a diploid status was observed in one or in both biopsied blastomeres. In 10 out of the 161 (6.2%) embryos a heterogeneity among the two biopsied blastomeres was found: a diploid nucleus in one blastomere and a non-diploid pattern or binuclear status in the other. In the remaining 53 (32.9%) out of 161 diagnosed embryos, the biopsied blastomeres were abnormal. The embryos that were not transferred or frozen were further analysed. When two sex chromosomes and two autosomes were present in the biopsied blastomere, the sex determination of the biopsied blastomere was never in conflict with the sex determination in the rest of the embryo. Furthermore, if the biopsied cell was diagnosed as abnormal (triploid, aneuploid, chaotic) the embryo was indeed completely abnormal or at least mosaic. A FISH error could not be excluded in two embryos (1.2%); however, a wrong gender determination did not result from this.

Preimplantation genetic diagnosis of Marfan syndrome with the use of fluorescent polymerase chain reaction and the Automated Laser Fluorescence DNA Sequencer.

OBJECTIVE: To develop and apply clinical preimplantation genetic diagnosis (PGD) for Marfan syndrome. DESIGN: Case report. SETTING: Centers for medical genetics and reproductive medicine in university hospitals. PATIENT(S): One couple in which the husband was affected with Marfan syndrome. INTERVENTION(S): The couple underwent three intracytoplasmic sperm injection cycles. MAIN OUTCOME MEASURE(S): The correct diagnosis was obtained for embryos in three PGD cycles. RESULT(S): Although all the PGD cycles were followed by ET, no pregnancy ensued. CONCLUSION(S): This assay can provide a reliable and accurate preimplantation diagnosis of Marfan syndrome.

Preimplantation diagnosis for fragile X syndrome based on the detection of the non-expanded paternal and maternal CGG.

Fragile X syndrome is the most common monogenic cause of mental retardation in boys. It is always characterized clinically by moderate mental retardation and often by a long face with large everted ears and macro-orchidism. The causal mutation is an expansion of a CGG triplet repeat in a 5' exon of the FMR-1 gene in Xq27.3. We report here for the first time a method for preimplantation genetic diagnosis (PGD) for fragile X syndrome based on the amplification of the CGG triplet in the normal allele. Our candidate-patient population, as well as two clinical preimplantation genetic diagnosis (PGD) cycles which led to a pregnancy with an unaffected fetus, are presented in this paper.

Successful preimplantation genetic diagnosis is related to the number of available cumulus-oocyte complexes.

The inheritance pattern of monogenic inheritable disorders influences the proportion of unaffected embryos after preimplantation genetic diagnosis (PGD). We aimed to investigate the influence of the number of cumulus-oocyte complexes (COC) on the outcome after PGD. Eighty-four cycles of 47 couples were included in our analysis. All couples were at risk of transmitting autosomal recessive, autosomal dominant, X-linked single gene disorders or sexaneuploidies to their offspring. One PGD cycle was carried out for a Yq-deletion of the man. The correlation between the numbers of COC and biopsied embryos and between the numbers of COC and unaffected embryos was highly significant (P <0.05). A pregnancy occurred in 15 cycles and a minimum of six COC were needed to achieve a pregnancy. Thirteen pregnancies were observed in cycles with at least 9 COC. The transfer rate and number of transferred embryos per cycle in the subgroups with <9 COC and > or =9 COC were significantly higher in the latter. Although pregnancy rates did not differ significantly between the two subgroups (probably due to the low number of pregnancies), our data indicate that it is justifiable to cancel PGD cycles in which it is expected that <6 COC will be retrieved and that the couple should be informed about the poor prognosis if <9 COC are retrieved.

Pregnancy after preimplantation genetic diagnosis for Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth (CMT) disease type 1A is an autosomal dominant peripheral neuropathy characterized by slow progressive distal muscle wasting and weakness, and decreased nerve conduction velocities. Most CMT1A cases (>98%) are caused by a duplication of a 1.5 Mb region on the short arm of chromosome 17 containing the PMP22 gene. A couple with a previous history of CMT followed by termination of pregnancy was referred to our centre for preimplantation genetic diagnosis (PGD). The husband carries the CMT1A duplication which can be detected by polymerase chain reaction (PCR) analysis using polymorphic (CA)n markers localized within the duplication. PCR amplification of genomic DNA of the parents-to-be with one of the two primers labelled with fluorescein, followed by automated laser fluorescence (ALF) gel electrophoresis of the amplified fragments allows the distinction between both genotypes. Embryos obtained after intracytoplasmic sperm injection (ICSI) were evaluated for the presence of the normal allele of the father. PCR with single Epstein-Barr virus-transformed lymphoblasts and blastomeres resulted in 91.4 and 93.5% amplification efficiency respectively, whereas none of the blank controls gave a positive signal. Allele drop-out (ADO) was observed in eight out of 32 lymphoblasts (25%) or in five out of 21 blastomeres (23.8%). However, within this set-up ADO will never lead to transfer of an affected embryo. A first ICSI-PGD cycle did not result in embryo transfer for the patient. A second cycle involved 10 mature oocytes of which eight were fertilized, resulting in five embryos for biopsy. Two unaffected embryos were available for transfer and resulted in a singleton pregnancy. The genotype of the fetus has been confirmed healthy by chorionic villus sampling.

Fluorescent PCR and automated fragment analysis for the clinical application of preimplantation genetic diagnosis of myotonic dystrophy (Steinert's disease).

Myotonic dystrophy (DM), or Steinert's disease, is an autosomal dominant disease characterized by myotonia, muscular weakness and atrophy, as well as lens opacities, cardiomyopathy and mild endocrine changes. The gene for DM located on 19q contains a triplet repeat at the 3' end of the gene. In DM patients, this repeat is found to be expanded. We have previously described a preimplantation genetic diagnosis (PGD) for DM using polymerase chain reaction (PCR) followed by conventional analysis on ethidium bromide-stained gels. The major drawback of this system was that allelic dropout occurred in >20% of the cells, leading to the loss of healthy embryos for transfer. To resolve this problem, we developed a PGD for DM using fluorescent PCR followed by fragment analysis on an automated DNA sequencer and made a comparison between the conventional PCR described earlier and fluorescent PCR, which turned out to be superior in accuracy and efficiency. Three PGD cycles were performed using fluorescent PCR and are described here.

Do we treat the male or his gamete?

The history of the male infertility patient is of utmost value. A physical examination is mandatory when psychosexual and ejaculatory dysfunction and male accessory gland infection are suspected, and even in the presence of azoospermia. It is also advisable to perform a physical examination to exclude the presence of testicular tumours. The diagnostic assessment includes sperm analysis, history, physical examination, the Valsalva manoeuvre, Doppler, ultrasonography, hormonal serum measurements, evaluation of testicular volume by orchidometry and evaluation of testicular consistency by palpation. The diagnosis of male infertility is descriptive and determination of true causality is almost non-existent. For decades, various therapies have been proposed to improve sperm parameters in cases of male factor infertility. Administration of anti-oestrogens and androgens is ineffective. No peer-review data are available to demonstrate the benefit of the use of intrauterine insemination or the correction of varicocele. Classic in-vitro fertilization is to some extent a solution for male factor infertility; however, the two-pronuclear fertilization rate for patients with impaired semen samples is significantly lower than that for patients with non-male indications. Conventional treatment for male factor infertility has little value and has been revised and abandoned. Intracytoplasmic sperm injection is an effective treatment, even for cases of extreme oligoasthenoteratozoospermia. It has to be considered the method of choice and should replace ineffective conventional therapies.