玻璃化冷冻法冻融经植入前遗传学诊断后囊胚的应用初探

目的:探讨玻璃化冷冻技术冻融经卵裂球活检后囊胚的可行性。方法:将活检后剩余的可移植囊胚用玻璃化冷冻保存,并在冷冻前人工皱缩囊胚腔,在需要移植时予以解冻囊胚进行移植。结果:24例共进行24个活检周期,活检了159个胚胎,活检后胚胎囊胚形成率60.38%(96/159)。有17个周期共移植26枚新鲜可用囊胚,成功种植13个(50.0%),11例获得临床妊娠(64.71%),7个周期因无可移植胚胎或卵巢过度刺激等因素而取消移植。10例患者(10个周期)有30个可移植囊胚进行了玻璃化冷冻保存,其中6例患者因未成功生育要求解冻其囊胚进行移植。共解冻8枚囊胚,全部存活并移植,5例获单胎妊娠;2例已分娩正常婴儿,3例继续妊娠中。结论:玻璃化冷冻技术结合人工皱缩囊胚腔能冷冻保存经卵裂球活检后的囊胚。     

Dilemmas encountered with preimplantation diagnosis of aneuploidy in human embryos

BACKGROUND: An increased embryo aneuploidy rate is associated with advancing maternal age. Preimplantation genetic diagnosis (PGD) using fluorescence in situ hybridisation (FISH) coupled with in vitro fertilisation (IVF)/embryo biopsy provides a powerful tool to improve the take home baby rates for this poor prognostic group. AIM: To report the preliminary findings of a PGD study for aneuploidy screening and to discuss the dilemmas encountered. METHODS: Preimplantation genetic diagnosis was offered in egg pick up-PGD and frozen embryo transfer-PGD cycles. Embryo biopsy was carried out on day 3 and FISH was used to detect chromosomal abnormalities. RESULTS: The outcome of 75 patients, 100 treatment cycles; 62 egg pick up-PGD and 38 frozen embryo transfer-PGD are presented. The embryo biopsy rate, blastomere survival, presence of nuclei and successful FISH rates for egg pick-up and frozen embryo transfer cycles were similar giving a chromosomal abnormality rate of 57.5 and 51.2% for the respective treatment group. The positive pregnancy, clinical pregnancy and implantation rates were, for egg pick up-PGD 22.7, 13.6 and 21.1% and for frozen embryo transfer-PGD 13.8, 10.3 and 10.0%, respectively. CONCLUSIONS: Preimplantation genetic diagnosis coupled with IVF treatment seems to give satisfactory pregnancy rates. The high embryo aneuploidy rates, chromosomal mosaicism and other issues have presented significant ethical and management dilemmas for our physicians and patients alike. These issues highlight the importance of skillful pretreatment counselling for patients considering PGD.     

Blastocyst trophectoderm biopsy and preimplantation genetic diagnosis for familial monogenic disorders and chromosomal translocations

OBJECTIVE: Modern in vitro fertilization practices involve transfer of embryos as blastocysts, when anabolic metabolism is well established and pregnancy rates can be maintained while transferring embryos singly to avoid multiple pregnancies. Embryo biopsy for preimplantation genetic diagnosis (PGD), however, is generally performed on day 3, when the embryo comprises just 6 to 8 cells, one or two of which are removed for testing. Implantation rates and clinical pregnancy rates have remained relatively low and a harmful effect from losing one or more cells from such early embryos has not been excluded. METHODS: We performed a sequential study involving 399 egg retrievals and 1879 embryo biopsies for patients undergoing PGD to avoid a serious monogenic disease or an unbalanced chromosomal translocation. We compared implantation and viable pregnancy rates after biopsies taken on day 3 (cleavage-stage biopsy) with biopsies delayed until day 5 or 6, when the embryo is a blastocyst and 5 or more cells can be sampled from the trophectoderm while the inner cell mass, from which the fetus develops, remains intact. All embryos were transferred as blastocysts. RESULTS: Despite fewer blastocysts than cleavage embryos biopsied and tested (3.6 compared to 6.6), implantation rates per embryo transferred were 43.4% if biopsied at the blastocyst stage and 25.6% if biopsied at the cleavage stage (P < 0.01), with ongoing or live-birth pregnancy rates per egg retrieval of 34.2% (average transfer number 1.1) for blastocyst biopsies and 25.5% (transfer number 1.6) for cleavage stage biopsies (P < 0.05, 1-tailed). The multiple pregnancy rate for monogenic disease exclusion fell from 16.7% to 2% (P = 0.04, 1-tailed). CONCLUSIONS: For exclusion of genetic disease, day 5-6 blastocyst-stage biopsies are more likely to be followed by implantation and singleton births than is the case after PGD performed on day 3.     

The use of preimplantation genetic diagnosis in sex selection for family balancing in India

This paper describes the use of preimplantation genetic diagnosis (PGD) in sexing embryos for family balancing in a private IVF clinic in India from April 1999 to April 2001. Embryos were biopsied and analysed on day 3, cultured in sequential media and then transferred on day 4 or day 5 after morphological selection of the best embryos. From a total of 42 cycles started, 14 clinical pregnancies and nine live births have been achieved so far, with five ongoing pregnancies. The benefits of delayed transfer 24-48 h after the embryo biopsy are that PGD centres could use the extra time available to confirm the diagnosis or introduce additional diagnostic tests for the same embryo. The selection of blastocysts for transfer should also permit the transfer of fewer embryos, thus reducing the risk of multiple gestations and increasing the pregnancy rate as a consequence of the expected higher implantation rate. This is the first report of the use of PGD in sex selection for family balancing in India, where couples place a premium on having baby boys, and the social and ethical aspects of the use of this technology in this setting are briefly discussed.     

Transfer of frozen-thawed blastocysts that underwent quarter laser-assisted hatching at the day 3 cleaving stage before freezing

OBJECTIVE: To examine the pregnancy potential of frozen-thawed blastocysts that underwent quarter laser-assisted hatching (AH) at the cleaving stage before freezing and to compare clinical and embryo characteristics between the groups that succeeded in and failed to achieve pregnancy. DESIGN: Prospective observational study. PATIENT(S): Thirty-four of 112 patients with frozen blastocysts requiring transfer of thawed embryos between January 2000 and December 2001. SETTING: Assisted reproductive technology unit in a tertiary medical center. INTERVENTION(S): Embryos of patients undergoing blastocyst transfers routinely underwent quarter AH using a nontouch 1.48-microm diode laser. Blastocysts not transferred were cryopreserved using a six-step freezing protocol with glycerol as the cryoprotectant. MAIN OUTCOME MEASURE(S): Postthaw embryo survival and zona pellucida (ZP) maintenance, implantation rate, and clinical pregnancy rate per transfer of thawed blastocysts. RESULT(S): A total of 118 frozen blastocysts was thawed. Of these, 89 (75.4%) embryos survived and were transferred in 35 cycles. Assisted hatching-manipulated ZP tolerated the freeze-thaw procedures without shape distortion in surviving embryos. Eleven (31.4%) clinical pregnancies with 15 intrauterine gestational sacs occurred, for an implantation rate of 16.9%. Those who succeeded in pregnancy tended to have more embryos available before freezing, more original blastocysts of good quality for freezing, and more surviving blastocysts after thawing for transfer than did those who failed to achieve pregnancy. CONCLUSION(S): An acceptable clinical pregnancy rate was obtained from transfer of thawed blastocysts that underwent laser AH on the ZP at the day 3 cleaving embryo stage in fresh cycles in selected patients. Embryo characteristics before freezing played major roles in determining implantation potential of thawed embryos.     

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.     

Pregnancy after polar body biopsy and freezing and thawing of human embryos

OBJECTIVE: To evaluate the outcome of frozen-thawed ET using embryos previously biopsied for preimplantation genetic diagnosis during a fresh ET cycle. DESIGN: Prospective evaluation. SETTING: Assisted reproductive biology program. PATIENT(s): A 31-year-old, G4, P1, TAB1, SAB2 carrier of a balanced RT 45,XX der(14;21)(q10;q10) translocation. INTERVENTION(s): Preimplantation genetic diagnosis by polar body biopsy. Excess embryos were frozen using the one-step method and then thawed. MAIN OUTCOME MEASURE(s): Embryo survival after thawing and subsequent pregnancy outcome. RESULT(s): Among the 32 mature oocytes, the results of fluorescence in situ hybridization were available for 25 polar bodies. Eleven were unbalanced, 10 were normal (8 fertilized), and 4 were balanced (3 fertilized) for the fresh IVF cycle. Two normal embryos were transferred. Four normal and 3 balanced embryos were cryopreserved. A chemical pregnancy resulted. Four months later, the 7 cryopreserved embryos were thawed; 2 survived (1 balanced and 1 normal) and were transferred. An ongoing pregnancy resulted, and a normal (46,XX) female was delivered. CONCLUSION(s): Freezing and thawing of biopsied embryos resulted in a low survival rate. However, this should not be a deterrent to the cryopreservation of extra chromosomally normal embryos because the embryos that do survive are able to implant.     

多囊卵巢综合征妇女体外成熟卵母细胞胚胎慢速冷冻复苏移植结局分析

目的评价未成熟卵母细胞体外成熟（IVM）后形成的卵裂期胚胎经慢速冷冻一解冻后的发育能力。方法将2006年1月至2010年12月北京大学第三医院因多囊卵巢综合征（PCOS）合并不孕症行卵裂期胚胎复苏移植的385例患者分为两组：复苏胚胎来源于体外成熟的卵母细胞组（IVM组,46例）和复苏胚胎来源于常规体内成熟的卵母细胞组（IVF组,339例）。采用慢冻速溶法解冻移植后比较两组患者的临床结局。结果IVM组复苏胚胎243枚,复苏后存活162枚,复苏率为66．67％;IVF组复苏胚胎1605枚,复苏后存活1082枚,复苏率为67．41％,两组比较,差异无统计学意义（P〉0．05）。IVM组患者的临床妊娠率和着床率分别为19．30％（11／57）和10．61％（14／132）,明显低于IvF组临床妊娠率（45．45％,175／385）和着床率（26．14％,240／918;P均〈O．05）。结论体外成熟卵母细胞发育形成的卵裂期胚胎慢速冷冻后临床结局欠佳,可能与冻融前胚胎自身的发育潜力有关。     

Successful application of the strategy of blastocyst biopsy, vitrification, whole genome amplification, and thawed embryo transfer for preimplantation genetic diagnosis of neurofibromatosis type 1

ObjectivePreimplantation genetic diagnosis (PGD) offers an alternative for women to carry an unaffected fetus risk of hereditary diseases. Trophectoderm biopsy may provide more cells for accurate diagnosis. However, the time allowed for transportation of the specimens to the laboratory and performance of molecular diagnosis is limited. We designed a PGD program of trophectoderm biopsy, vitrification of blastocysts, whole genome amplification (WGA), double confirmatory genotypings, and thawed embryo transfer.Case ReportWe conducted this strategy for a woman of familial neurofibromatosis type I (NF-1). She had a genotype of heterozygous c.6709C>T mutation of NF1 gene. Trophectoderm biopsies were performed on 13 blastocysts. Then, individual blastocyst was vitrified. WGA was performed for the samples, followed by genotypings with both real-time polymerase chain reaction and sequencing. Eight embryos were diagnosed as unaffected, four were affected, and one was inconclusive because of allele drop-out. In the next cycle, two unaffected blastocysts were thawed and transferred, that resulted in a singleton pregnancy. The pregnancy was confirmed as unaffected by means of chorionic villi sampling.ConclusionWe first demonstrate successful application of blastocyst biopsy, vitrification, WGA, and thawed embryo transfer for PGD of a monogenic disease. Vitrification of blastocysts after biopsy permits sufficient time for shipment of samples and operation of molecular diagnosis.     

Preimplantation genetic diagnosis: the earliest form of prenatal diagnosis

Preimplantation genetic diagnosis (PGD) can provide genetic information on embryos obtained through in vitro fertilization (IVF), allowing implantation of embryos identified as unaffected with a given genetic or chromosomal disorder. With the availability of increasingly sophisticated genetic testing, its use has advanced from the selection of female embryos for the prevention of X-linked genetic diseases to testing for single gene disorders via PCR. Recently, PGD has also been used in the setting of assisted reproductive technology to select for chromosomally normal embryos in an effort to increase the rates of implantation and successful pregnancy. As the number of patients undergoing IVF increases, the indications for its use broadens, and more mutations underlying genetic disorders are identified, PGD is becoming more widespread. As this evolution continues, recognition of the limitations of PGD, as well as ethical concerns regarding use and misuse of this technology, need to be considered by patients, clinicians, and policy makers.     

Clinical management of in vitro fertilization with preimplantation genetic diagnosis

Patients who undergo in vitro fertilization (IVF) because of preimplantation genetic diagnosis (PGD) require different clinical management than those who come in because of infertility alone. PGD adds a "fourth dimension" to the emotional aspect of a patients' assisted reproductive technology treatment. It significantly decreases the number of embryos available for transfer by 25 to 81%, and therefore ovarian stimulation for IVF with PGD should be tailored individually, taking into account patients' safety and estimated ovarian reserve. Recent studies showed that with increased number of oocytes retrieved, the higher the chance to have an embryo transfer and normal cryopreserved blastocysts. With adequate ovarian stimulation, there is no cutoff for the numbers of oocytes/embryos needed to start PGD with, especially for younger patients. Patient-friendly protocols, such as those based on gonadotropin-releasing hormone antagonist and vaginal progesterone support may be used. Elective single embryo transfer and blastocysts cryopreservation to avoid multiple pregnancies may be offered with PGD. The benefit of adding preimplantation genetic screening to IVF treatment is still controversial, and evidence-based data on 24-chromosome testing of polar bodies or trophectoderm is needed before it may be implemented into routine patient care.This review discusses the clinical management of IVF with PGD based on the best available data and my personal clinical experience as a reproductive specialist with >1000 IVF/intracytoplasmic sperm injection-PGD cycles. The information provided here will assist reproductive specialists, nurses, geneticists, genetic counselors, and embryologists to better counsel and treat couples who wish to conceive with a healthy child through IVF with PGD. It is time for PGD to be viewed as a modern modality of preventive medicine. As such, it should be incorporated into national health-care systems and be covered by medical insurance.     

Successful dizygotic twin pregnancy after recryopreservation by vitrification of human expanded blastocysts developed from frozen cleaved embryos on day 6: a case report

BACKGROUND: Vitrification has evolved into an established technique for cryopreservation of human blastocysts. However, it is still unclear whether the blastocysts developed from frozen embryos can be cryopreserved a second time by vitrification for further embryo transfer. CASE: A 31-year-old woman underwent a long-treatment protocol for ovarian stimulation. Twenty-seven mature oocytes were obtained, and 21 were fertilized with intracytoplasmic sperm injection. On day 3, 2 cleaved embryos were transferred, but no implantation occurred. The remaining 19 embryos were cryopreserved with the slow freezing method. Three months after oocyte retrieval, 5 frozen day 3 embryos were thawed, the surviving 2 were transferred, but no implantation occurred. Six months after oocyte retrieval, the remaining 14 frozen day 3 embryos were thawed and the surviving 12 cultured. On day 5, 2 embryos reached the expanded blastocyst stage and were transferred, but no implantation occurred. On day 6, 5 of the nontransferred embryos became expanded blastocysts and were cryopreserved again by vitrification. Eight months after oocyte retrieval, 2 recryopreserved day 6 blastocysts were warmed and transferred. Implantation resulted in a dizygotic twin pregnancy. The pregnancy resulted in delivery of normal, healthy male and female infants weighing 2,155 and 2,590 g at birth, at 36 weeks of gestation. CONCLUSION: The blastocysts developed from frozen embryos on day 6 can be recryopreserved by vitrification and have pregnancy potential after warming.     

Preimplantation genetic diagnosis significantly improves the pregnancy outcome of translocation carriers with a history of recurrent miscarriage and unsuccessful pregnancies

Preimplantation genetic diagnosis (PGD) for translocations has been shown to significantly reduce the risk of recurrent miscarriage, but because the majority of embryos produced are unbalanced, pregnancy rate is relatively low since 20% or more cycles have no normal or balanced embryos to transfer. The purpose of this study was to evaluate whether PGD could improve pregnancy outcome in translocation carriers with a history of two or more consecutive miscarriages and no live births. PGD for translocations was offered to translocation carriers with two or more previous miscarriages (average 3.5) and no live births (0/117 pregnancies) using a combination of distal and proximal probes to the breakpoints. After PGD, only 18.3% of embryos were normal or balanced. Only 5.3% of pregnancies were lost after PGD compared with 100% before PGD (P < 0.001). The cumulative pregnancy rate was 57.6% and the cumulative ongoing pregnancy rate was 54.5% in the short period of time of 1.24 IVF cycles, or 46.3% and 43.9% respectively per cycle. In conclusion, PGD significantly reduced losses and increased the number of viable pregnancies (P < 0.001). IVF plus PGD are a faster method of conceiving a live child than natural conception, at least for translocation carriers with recurrent miscarriages and no previous live births.     

Blastocyst culture: toward single embryo transfers

The routine culture and transfer of viable human blastocysts has been made possible by the development of sequential culture media, formulated to account for the changes in nutrient requirements of the embryo as it develops and differentiates. Resultant implantation rates of blastocysts transferred on day 5 are significantly higher than those obtained by the transfer of cleavage stage embryos transferred on day 2 or day 3 within the same programme. As a direct result of this increase in implantation rate, fewer blastocysts than cleavage stage embryos need to be transferred to obtain acceptable pregnancy rates, thereby reducing the incidence of multiple gestations. Blastocysts developed in sequential culture media are readily cryopreserved. The efficiency of in vitro fertilization (IVF) in a general patient population can be calculated using a model that takes into account the number of embryos transferred and cryopreserved, together with their respective implantation rates. Blastocyst transfer is associated with about a 20% increase in the efficiency of IVF compared with the transfer of cleavage stage embryos on day 3. The development of a suitable scoring system has enabled identification of those blastocysts with the highest developmental potential (70% implantation rate). The culmination of this work should be the move to the transfer of a single blastocyst for a significant number of patients.     

Blastocyst culture: Toward single embryo transfers

The routine culture and transfer of viable human blastocysts has been made possible by the development of sequential culture media, formulated to account for the changes in nutrient requirements of the embryo as it develops and differentiates. Resultant implantation rates of blastocysts transferred on day 5 are significantly higher than those obtained by the transfer of cleavage stage embryos transferred on day 2 or day 3 within the same programme. As a direct result of this increase in implantation rate, fewer blastocysts than cleavage stage embryos need to be transferred to obtain acceptable pregnancy rates, thereby reducing the incidence of multiple gestations. Blastocysts developed in sequential culture media are readily cryopreserved. The efficiency of in vitro fertilization (IVF) in a general patient population can be calculated using a model that takes into account the number of embryos transferred and cryopreserved, together with their respective implantation rates. Blastocyst transfer is associated with about a 20% increase in the efficiency of IVF compared with the transfer of cleavage stage embryos on day 3. The development of a suitable scoring system has enabled identification of those blastocysts with the highest developmental potential (70% implantation rate). The culmination of this work should be the move to the transfer of a single blastocyst for a significant number of patients.     

Initial experience with extended culture and blastocyst transfer of cryopreserved embryos

Objective: Our purpose was to evaluate the viability and transfer efficiency of cryopreserved embryos allowed to develop into blastocysts in extended culture for in vitro fertilization. Study Design: The embryos for in vitro fertilization that had been cryopreserved at either 2 PN (pronuclear) or cleaving stage (day 1-3) were thawed and cultured for uterine transfer on day 5. Outcome for day 5 embryo transfer was prospectively compared with previous outcomes from embryos transferred on day 2 or 3. Results: For embryos thawed and transferred on day 2 or 3 (n = 99), the pregnancy rate was 33%, the implantation rate per embryo transferred was 15.2%, and the rate of multiple gestations was 42.4% (14/33) with 35.7% of pregnancies having ≥3 gestational sacs. For extended culture embryos transferred on day 5 (n = 25), the pregnancy rate was 36%, the implantation rate per embryo transferred was 16.7%, and the rate of multiple gestations was 33.3% (3/9) with all of these being twins. For embryo transfers performed on day 5 in which only blastocysts were transferred (n = 9), the pregnancy rate was 66.7%, the implantation rate per blastocyst was 44.4% (greater than the rate for the day 2 or 3 embryos, P = .0043), and the rate of multiple gestations was 33.3% (2/6) with all of these being twins. In extended culture 29.8% of cryopreserved embryos progressed to the blastocyst stage. In this series 4 subjects (15.4%) did not have blastocysts by day 5. Conclusion: Acceptable pregnancy rates can be obtained from cryopreserved embryos cultured to the blastocyst stage with a significantly higher implantation rate. Transfer of embryos that have “self-selected” to blastocysts results in reduced risk of higher-order (>2) multiple gestations, because only 1 or 2 embryos are transferred. (Am J Obstet Gynecol 1999;180:1472-4.)     

Embryo pooling: a promising strategy for managing insufficient number of embryos in preimplantation genetic diagnosis

This retrospective study evaluated the embryo pooling strategy for managing insufficient number of embryos in preimplantation genetic diagnosis (PGD) through serial vitrification of cleavage-stage embryos from consecutive cycles, and simultaneous blastocysts biopsy in combination with blastocysts obtained in ultimate fresh cycle. A retrospective analysis of the cumulative pregnancy rate of 68 patients underwent cleavage-stage embryos accumulation (Embryo Pooling Group) and 94 patients underwent one stimulation cycle (Control Group) over a 2-year period were conducted. The blastocyst formation rate was comparable between the consecutive cycles and the ultimate cycle in embryo pooling group (56.0 versus 62.0%, p?=?.078). No significant difference existed between twice-vitrified and once-vitrified warmed blastocysts with respect to implantation rate (50.8 versus 46.3%, p?=?.658). The implantation rate and cumulative pregnancy rate of embryo pooling group were 49.0 and 67.6%, respectively, which were statistically comparable to the corresponding values of 48.9 and 73.4% obtained in control group. Our study suggests that in patients undergoing ICSI-PGD who do not reach enough embryos in a single stimulation cycle, pooling embryos from consecutive ovarian stimulation cycles is a promising strategy, which can render a cumulative pregnancy rate comparable to those patients who only require one stimulation cycle.     

Improved implantation after preimplantation genetic diagnosis of aneuploidy

The objective of this study was to assess the improvement in implantation rates after preimplantation genetic diagnosis (PGD) of numerical abnormalities for the sole indication of advanced maternal age when compared with a control group. Each PGD patient was matched to a control patient according to several parameters prior to obtaining pregnancy results. The diagnosis was based on the analysis of chromosomes X, Y, 13, 15, 16, 18, 21 and 22 plus a ninth probe (1, 7, 14 or 17) on a single cell per embryo. The results were also analysed in relation to the previous number of IVF cycles and the number of dipronucleated zygotes obtained, when replacing presumptively chromosomally normal embryos on day 4 of development. It was found that women of advanced reproductive age (average age 40 years) had a higher implantation rate (18%) than their matched controls treated with standard IVF (11%) (P < 0.05). This increase was not observed in patients with two or more previous IVF cycles or patients with fewer than eight zygotes. Patients with eight or more 2PN zygotes and one or no previous cycles showed the greatest improvement in implantation rate, from 8.8% in controls to 19.2% in the PGD group (average age 40 years) (P < 0.025).     

Preimplantation diagnosis for p53 tumour suppressor gene mutations

Preimplantation genetic diagnosis (PGD) was introduced for high-risk couples to avoid establishing affected pregnancies potentially requiring termination following prenatal diagnosis. This opens the possibility for PGD for late onset disorders with genetic predisposition, including inherited cancer predisposition, because only embryos free from the predisposing gene may be transferred back to the patient, with no potential risk for pregnancy termination. PGD was performed for two couples, one with maternally and one with paternally derived p53 tumour-suppressor mutations, 902insC in exon 8 and G524A in exon 5, respectively. This involved a standard IVF protocol, allowing oocytes or embryos to be tested prior to their transfer back to uterus. Maternal mutation was tested by sequential PCR analysis of the first and second polar bodies, removed following maturation and fertilization of oocytes, while paternal mutation analysis required embryo biopsy at the cleavage stage. To avoid misdiagnosis due to allele drop out, multiplex nested PCR was applied, involving p53 mutation analysis simultaneously with the linked short tandem repeats in intron 1. Of 10 oocytes tested in two PGD cycles for 902insC mutation, four unaffected oocytes were pre-selected for transfer yielding no clinical pregnancy. Of 18 embryos analysed in two cycles for G524A mutation, seven mutation-free embryos were detected, two of which were transferred in each cycle, resulting in a singleton pregnancy and birth of a mutation-free child. This is the first PGD for inherited cancer predisposition determined by p53 tumour suppressor mutations, resulting in a clinical pregnancy and birth of a child free from inherited cancer predisposition.