JBRA Assist. Reprod. 2000;04(02):88-92
RELATO DE CASO
doi: 10.5935/1518-0557.2000.4.2.09
Blastocyst transfer after embryo biopsy and gender selection in a woman with fragile X premutation and diminished ovarian reserve
Transferência de blastocisto após biópsia e seleção em uma mulher com frágil X premutação e reserva ovariana diminuída
1Centro de Reprodução Humana Huntington-São Paulo
2Laboratório de Genética Humana, Instituto de Medicina Fetal e Genética Humana
3Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo
Abstract
Mutations in the fragile X mental retardation I (FMR I) gene are diagnosed in the amniotic fluid and chorionic villus sampling of individuais with full mutations and premutations. Transfer of 46,XX IVF generated embryos reduces the probability of FMR l gene full mutation. Carriers with premutation are at increased risk of premature ovarian failure. This report describes the results of 3 IVF attempts in a 38 year old woman with fragile X premutation and reduced ovarian reserve. After signing informed consents, the patient underwent ovulation induction using short Lupron protocol. A 3 colour DNA FISH labelling was performed in single blastomeres obtained at the 8-cell stage embryos with probe set specifically for chromosomes 18, X, andy All 8-cell embryos were biopsied and only blastocysts with XX complement were transferred. The mechanical impact of the biopsy did not appear to compromise blastocyst formation. Abdominal chorionic villus sampling (CVS) performed at 12 weeks gestation revealed a 46,XX foetal karyotype and normal alleles (2.8 kb unmethylated fragments) excluding premutation. The patient delivered a normal female infant via caesarean section who weighed 3.9 Kg. In conclusion, IVF generated female embryos reduce the probability af full fragile X mutation; nevertheless, prenatal diagnosis is still indispensable.
Key words: Fragile X, fragile X mental retardation 1 (FMRl) gene, FMRl gene mutation and premutation, fluorescence in situ hybridisation (FISH) of single blastomeres, blastocyst transfer.
Resumo
Mutações em genes X mental retardação I (FMRI) são diagnosticadas no líquido aminiótico e na amostra de vilo corial com muitas mutações e premutações. Das transferências de 46, XX, as FIV geralmente reduziram a probabilidade de FMRI gene com muita mutação. Carriers com premutação estão aumentando o risco de falência ovariana prematura. Este relato descreve o resultado de 3 tentativas de FIV em mulheres com 38 anos com frágil X premutação e reserva ovariana reduzida. Após consentimento informado e assinado a paciente foi submetida a estimulação ovariana usando protocolo de Lupron curto. As 3 etiquetas de coloração de FISH DNA foi realizada em blastômeros simples obtidos em estágio embrionário de 8 células com sonda específica para cromossômos 18, X e Y. Todas as 8 células embrionárias obtidas foram biopsiadas e somente blastocisto com XX complemento foram transferidos. O impacto mecânico da biópsia não parece comprometer a formação do blastocisto. Amostra de vilo corial abdominal (CVS) realizado na 12" semana de gestação revelaram 46 XX, cariótipo fetal e normal alelos. (2.8 Kb de fragmentos não metilados) excluindo premutação. A paciente teve uma criança normal do sexo feminino pesando 3.9 kg. Em conclusão FIV geralmente em embriões femininos reduzem a probabilidade de muita frágil X mutação, e diagnóstico pre-natal é ainda indispensável.
Unitermos: frágil X, frágil X retardação mental I (FMPl) gene, FMRI mutação gene e premutação, fluorescência de hibridação in situ (FISH) de blastômeros simples, tranferência de blastocisto.
Introduction
Fragile X is one of the most common causes of inherited mental retardation in humans (Hagerman et al., 1985; Winter, 1989). This X-linked dominant condition within complete penetrance is caused by mutations in the fragile X mental retardation 1 (FMR1) gene and direct DNA testing allows accurate diagnosis in the blood, amniotic fluid, and chorionic villus samples of individuals with both full. mutations and premutations (Yu et al, 1991; Dobkin et al, 1991; Warren and Ashley, 1995). Nevertheless, for many fragile X carriers and their families, abortion of an affected foetus is not an acceptable choice. Furthermore, the alternatives of oocyte donation, adoption, and not procreating comprise additional non-cherished options. The use of preimplantation genetic diagnosis (PGD) as the preferable reproductive identification of potentially compromised embryos in families affected by this X-linked disorder would surge as the elective course. However, the lack of diagnosing FMRl gene mutation in a single blastomere still represents a decisive shortcoming. Although the transfer of female pre-embryos reduces the probability of FMRl gene full mutation, there is Iimited number of IVF centres capable of providing embryo biopsy and sex chromosome identification. High expenses associated with this technological testing might become prohibitive to several communities in underdeveloped countries, besides the additional need of prenatal testing and genetic counselling. Furthermore, the establishment of either premature ovarian failure (POF) or impairments of ovarian reserve encountered in several young premutation carriers add to the complexities presented by these patients (Schwartz et al., 1994; Partington et al., 1996; Conway et al., 1998; Murray et al., 1999; Allingham-Hawkins et al., 1999). This report describes the results of three IVF attempts in a woman with fragile X premutation and reduced ovarian reserve. Sex chromosome analysis was achieved via fluorescence in situ hybridisation (FISH) of single blastomeres obtained from ali 8-cell biopsied pre-embryos. Extended in vitro culture to blastocyst stage was accomplished before uterine replacement of female embryos. The patient delivered a healthy term female infant fol lowing a normal chorionic villus cells DNA testing disclosing a 46,XX foetus without premutations.
Materiais and Methods
A 38 year old female carrier of FMRI gene premutation, GOPO, was initially referred for assisted conception and gender selection for the transfer of female embryos due to a family history of fragile X mental retardation. Two nephews bom from different sisters suffered from severe mental retardation related to this syndrome. The probability of mental retardation for the patient's offspring due to Fragile X was estimated to be in the neighboured of 29%. Past gynaecological and medical history was unremarkable. The patient was using an oral hormonal formulation as a contraceptive method. Physical examination was unremarkable. A work-up for the most commonly screened in fectious diseases was negative. Hormonal evaluation performed on menstrual cycle day 3 revealed a serum estradiol (E2) of 40.5 pg/ml and FSH egual to 17.9 mIU/ml. The average ovarian volumes measured by pelvic ultrasound were 2.5 and 2.0 cm3. The patient was euthyroid and euprolactinemic. Hysteroscopy disclosed a normal endometrial cavity. Her husband was a healthy 41- year-old man who never fathered a child but had a normal spermogram. The couple was carefully counselled by the staff of the 3 institutions about the benefits and limitations of the procedures before signing informed consents. The patient underwent three cycles of ovulation induction (01) using short protocols. In the first and attempt, Lupron (Leuprolide acetate, Abbot Lab, North Chicago, IL, USA) was used for two days and thereafter it was followed by the association of 150 IU of uFSH (Metrodin, Serono Lab, São Paulo, Brazil) and 150 IU of hMG (Pergonal, Serono Lab, São Paulo, Brazil). Ten oocytes were harvested, five oocytes fertilised normally and two cleaving pre-embryos were biopsied. PGO revealed one embryo with normal complements for chromosome 18, X, and Y. In the second embryo, four signals for chromosome X were found and embryo transfer was not performed. The second OI was accomplished with 225 IU of uFSH following a short GnRHa protocol resulting in eight oocytes. Seven pronucleate oocytes developed into five pre-embryos, which were biopsied at the 8-cell stage, and one was noted to be damaged. Three embryos with paired X chromosomes were transferred at early blastocyst stage on the fifth day post-retrieval, while a trisomy 18 embryo was not transferred. Pregnancy did not ensue. Before the last trial, baseline day 2 hormonal evaluation revealed serum FSH of 8.5 mIU/ml and E2 of 68. 1 pg/ml. Similar 01 protocol was used resulting in the retrieval oftwelve oocytes and eleven pre-embryos. Five pre-embryos developed to the 8-cell stage and were biopsied. Two pre-embryos showed only one signal for chromosome X, one embryo did neither reveal signals for the sex chromosomes, and the remaining two embryos had a normal complementary X chromosomes. The latter two embryos were transferred as fully expanded blastocysts. A normally implanted singleton pregnancy was established. Abdominal chorionic villus sampling carried out at 12 weeks gestation revealed trophoblastic cells with a 46,XX karyotype after Giemsa staining and G-banding. A three-dimensional morphologic ultrasound at 20 weeks of gestation was normal. The patient delivered a normal full-term female weighing 3,970 gm with Apgar scores of 9 and 10, at the first and fifth minutes, respectively, via caesarean section.
IVF culture
Oocyte retrieval, in vitro fertilisation and culture procedures were accomplished as previously reported (Alves da Motta et al., 1998). Preimplantation one and blastocyst media were used to support blastocyst growth.
Embryo biopsy and FISH Analysis
Cleavage stage embryo biopsy and blastomere fixation procedures were carried out as described (Gianaroli et al., 1999). A three colour DNA FISH labelling was carried out from single blastomeres obtained at the 8-cell stage embryos with probe set specifically for chromosomes 18, X, and Y from Vysis Aneuvysion (Downers Grove, IL, USA). Hybridisation occurred in a dark moist chamber at 37°C for a period of six hours after denaturing in formamide at 73° C and dehydration. Thereafter, the slides were individually washed, first at 73°C in O.4xssc for 2 minutes, which was followed by one minute wash at room temperature in 0.2 x ssc. The slides were mounted in DAPI II counterstain and read in a microscope with fluorescence capability. The capture of the images was carried out in a Zeiss Axioskpe microscope coupled to a CCD camera and controlled by ISIS Metasystems software. Figure I shows the PGD hybridised blastomere of a tran sferred bl astocyst demon strating the signals corresponding to the pairs of chromosome 18 (agua) and X (green).
Figure 1. Hybridised blastomere of a biopsied eight-cell embryo transferred in the cycle that resulted in a normal pregnancy showing the FISH signals for the X chromosomes in green and for chromosome 18 in aqua.
DNA Analysis (patient)
DNA extracted from blood and chorionic villi was doubly digested with Ecoriieagi and probed with STB 12.3 as previously described (Vianna-Morgante et al, 1996). After Southem blotting, the patient was identified as a carrier of premutation due to the presence of a 3.1 kb unmethylated fragment. Normal alleles (2.8 kb unmethylated fragments) were found in the sample from the chorionic villi.
Discussion
This report highlights both the importance of a comprehensive team counselling and the scope of contemporary assisted reproductive treatment to women with fragile X premutation suffering from diminished ovarian reserve. This patient possessed small ovaries as assessed by transvaginal measurement of mean volumes (Lass et al., 1997) and exhibited an abnormal D3 serum FSH leveis (17.9 mIU/ml), findings that are in accord with diminished ovarian reserve. Therefore, higher cancellation rates, lower number of oocytes and embryos, and possibly a greater number of chromosomally abnormal embryos were to be anticipated. The flfst ovarian stimulation resulted in the collection of ten oocytes; nonetheless, only two pre-embryos cleaved to 8- cell stage with one presenting two pairs of X chromosomes and unfortunately the apparently normal blastocyst had XY complement and was not transferred. However, other variables such as culture conditions might have contributed to this initial outcome, the patient's ovarian reserve might have played a larger role. The results of second teial summarised by the creation of one abnormal embryo (25%) and the lack of pregnancy initiation favours the diagnosis of diminished reserve; although the transfer of three early blastocysts with XX complement clouds, in part, the contention of impeding ovarian failure. Whereas baseline serum FSH (8.5 mIU/ml) was normal during the initiation of the third IVF, mildly elevated serum E2 (68.1 pg/rnl) met the criteria for poor ovarian reserve as reported by Rosenwaks and Davis (Rosenwaks and Davis, 1994). In colloboration, B lack and associates (Black et al., 1995) showed that eight of nine premutation carriers experienced poor ovarian response to 01, even though only four of the patients had serum FSH levels in the perimenopausal range. Additional studies, including the report of the International Collaborative Premature Ovarian Failure in Fragile X study, have recently demonstrated an increased risk of diminished ovarian reserve and POF among women with fragile X premutation (Murray et al., 1999; Allingham-Hawkins et al., 1999; Marozzi et al, 2000).
It is also of relevance the fact that post-biopsied early and cavitated blastocysts were selected for embryo transfer. Including the results of the three IVF cycles, twelve preembryos developed to 8-cells (34.8%) and six went on to the blastocyst stage (26. 1 %) from 23 pronucleate oocytes. Although this percentage of blastocyst development falls short from the obtained with normal ovarian responders (Alves da Motta et al, 1998), one has to take into consideration that 41 .7% of biopsied pre-e.mbryos carried chromosomal abnormalities related exclusively to chromosomes X (four embryos) and 18 (one embryo). Moreover, to contemplate that some of the eleven arrested pre-embryos (47 .8%) presented similar and/or additional sex and autosomal aberrations would not be unfounded. Speculative inferences regarding these high embryonic X chromosome aneuploidies, despite that all originated from a single patient could lay on non-disjunction of the fragile X chromosome of ovarian origino Although the studies of Nielsen (1986) have not been validated, lymphocyte culture from fragile X cariers exhibited a large number of hyperdiploid cells. This mechanism of chromosome segregation, post-zygote events and father inheritance could additionally affect fertility potential in fragile X women with premutation (Marozzi et al, 2000).
Since the successful establishment of PGD for couples at risk for genetically detectable diseases (Handyside et al, 1990), embryo replacement has been carried out at the cleaving and morulae stages (Handyside et al, 1990; Munné et al, 1998; Gianaroli et al, 1999). With the development of more suitable culture media to promote in vitro human blastocyst growth, it has been observed embryonic "self-election", limited highorder multiple pregnancies and superior implantation rates due to better synchronisation between embryo implantation capacity and uterine receptivity (Alves da Motta et al, 1998). Since blastocyst growth may take an additional 24-36 hours after reaching morulae, an additional time is perrnitted to the Molecular Biology laboratory review any questions or problems that may arise during the course of the procedure. In addition, it is of interest the fact that the mechanical impact caused by the biopsy for the removal of a blastomere appeared that it did not compromise blastocyst formation. Further, extending in vitro culture to transfer blastocysts seemed to be a beneficial choice since only one embryo reaching the blastocyst stage had an abnormal chromosomal complement. These findings are also in accord with the notion of in vitro embryonic self-selection and could be useful or the management of embryo transfer.
Taken together, embryonic gender selection contributes to reduce the probability of full expression of the fragile X mutation; nevertheless, prenatal diagnosis via chorionic villus to establish foetal karyotype and the presence of mutations of the FMRl gene carriers is indispensable. Prenatal diagnosis will become obsolete when pre-embryo testing for the fragile X syndrome becomes clinically available. In conclusion, this is the first publication to our knowledge that biopsied pre-embryos for gender selection were allowed to develop in vitro to the blastocyst stage before the transfer to the uterus of a woman carrier of fragile X premutation and with diminished ovarian reserve. A 46,XX foetus without premutations was diagnosed by chorionic villus cells DNA testing before the pregnancy progressed to the delivery of a healthy term female infant.
Acknowledgements:
The authors acknowledge the contributions of ali members of the IVF and nursing team of the Centro de Reprodução Humana Huntington-São Paulo, as well as the physicians and technicians of the Instituto de Medicina Fetal e Genética Humana. The authors also acknowledge the editorial work of Mr. Gustavo Serafini.
Winter R. M. - Fragile X mental retardation. Arch. Dis. Child., 64, 1223-1224; 1989.