Pre-implantation diagnosis

The access to human pre-implantation embryos that is afforded by procedures now developed for the treatment of infertility presents the possibility of very early prenatal diagnosis, before implantation in the uterus, of certain genetic diseases. Only the normal embryos would be replaced in the mother for initiation of implantation and pregnancy. Early experiments on a mouse model for Lesch-Nyhan syndrome (HPRT-deficiency) show that pre-implantation diagnosis of genetic disease is feasible.     

Pregnancy rates for grade 2 embryos following administration of synthetic GnRH at the time of transfer in embryo-recipient cattle

To succeed with pregnancy a bovine embryo must overcome the luteolytic mechanism and achieve recognition of pregnancy. It is understood that well developed embryos are more successful in achieving recognition of pregnancy than poorly developed ones. Attempts have been made to assist this recognition of pregnancy by utilising a number of hormonal supplements with varying levels of success. A study was undertaken to test the hypothesis that supplementation with synthetic GnRH at the time of transfer of Grade 2 embryos will enhance pregnancy rates in recipients receiving this category of embryo. Pairs of fresh and frozen Grade 2 embryos (n = 38) from 34 donor animals were allocated to the trial. Thirty eight pairs of recipients were used and one of each pair was randomly assigned to receive treatment on the day of embryo transfer (Day 7) with 5 ml of gonadorelin, containing a synthetic gonadotrophin releasing hormone, 0.1 mg/ml. Pregnancy diagnosis was carried out from 42 days post-transfer by either palpation per rectum or ultrasound scanning. Treatment, embryo processing, side of transfer, parity of recipient, breed of recipient and breed of donor dam showed no statistically significant effect on pregnancy rate. The overall pregnancy rate in this study was within commercially accepted limits for Grade 2 embryos at 38.2%. The pregnancy rates were 34.2 and 42.1% for the GnRH-treated and control groups, respectively and were not significantly different at P < 0.05. The failure of this treatment to improve pregnancy rates could be due to its effect being transitory therefore allowing subsequent pregnancy loss. The timing of the treatment post-transfer, treatment dose and potency of the GnRH analogue may also play a role in this. Further study is required to determine the hormonal or follicular status of prospective candidates for treatment before applying this as a whole herd regime.     

Setting up equine embryo gender determination by preimplantation genetic diagnosis in a commercial embryo transfer program

Preimplantation genetic diagnosis (PGD) allows identifying genetic traits in early embryos. Because in some equine breeds, like Polo Argentino, females are preferred to males for competition, PGD can be used to determine the gender of the embryo before transfer and thus allow the production of only female pregnancies. This procedure could have a great impact on commercial embryo production programs. The present study was conducted to adapt gender selection by PGD to a large-scale equine embryo transfer program. To achieve this, we studied (i) the effect on pregnancy rates of holding biopsied embryos for 7 to 10 hours in holding medium at 32 °C before transfer, (ii) the effect on pregnancy rates of using embryos of different sizes for biopsy, and (iii) the efficiency of amplification by heating biopsies before polymerase chain reaction. Equine embryos were classified by size (≤300, 300–1000, and >1000 μm), biopsied, and transferred 1 to 2 or 7 to 10 hours after flushing. Some of the biopsy samples obtained were incubated for 10 minutes at 95 °C and the rest remained untreated. Pregnancy rates were recorded at 25 days of gestation; fetal gender was determined using ultrasonography and compared with PGD results. Holding biopsied embryos for 7 to 10 hours before transfer produced pregnancy rates similar to those for biopsied embryos transferred within 2 hours (63% and 57%, respectively). These results did not differ from pregnancy rates of nonbiopsied embryos undergoing the same holding times (50% for 7–10 hours and 63% for 1–2 hours). Pregnancy rates for biopsied and nonbiopsied embryos did not differ between size groups or between biopsied and nonbiopsied embryos within the same size group (P > 0.05). Incubating biopsy samples for 10 minutes at 95 °C before polymerase chain reaction significantly increased the diagnosis rate (78.5% vs. 45.5% for treated and nontreated biopsy samples respectively). Gender determination using incubated biopsy samples matched the results obtained using ultrasonography in all pregnancies assessed (11/11, 100%); untreated biopsy samples were correctly diagnosed in 36 of 41 assessed pregnancies (87.8%), although the difference between treated and untreated biopsy samples was not significant. Our results demonstrated that biopsied embryos can remain in holding medium before being transferred, until gender diagnosis by PGD is complete (7–10 hours), without affecting pregnancy rates. This simplifies the management of an embryo transfer program willing to incorporate PGD for gender selection, by transferring only embryos of the desired sex. Embryo biopsy can be performed in a clinical setting on embryos of different sizes, without affecting their viability. Additionally, we showed that pretreating biopsy samples with a short incubation at 95 °C improved the overall efficiency of embryo sex determination.     

Preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) is an evolving technique that provides a practical alternative to prenatal diagnosis and termination of pregnancy for couples who are at substantial risk of transmitting a serious genetic disorder to their offspring. Samples for genetic testing are obtained from oocytes or cleaving embryos after in vitro fertilization. Only embryos that are shown to be free of the genetic disorders are made available for replacement in the uterus, in the hope of establishing a pregnancy. PGD has provided unique insights into aspects of reproductive genetics and early human development, but has also raised important new ethical issues about assisted human reproduction.     

Developmental Biology and Human Concerns

There is a large natural loss of human embryos in early gestation.Most conceptual losses occur before pregnancy has been diagnosedin the woman. It is now acknowledged that chromosomal aberrationsare the major etiologic agents responsible for spontaneous abortions.Fully 50 percent of naturally aborted embryos in the first trimesterhave an abnormal karyotype. Most of the chromosomal errors thathave been identified in abortuses are only rarely seen in livebirths.Natural in utero selection is relentless in eliminating 99 percentof the chromosomally abnormal conceptuses through spontaneousabortion. The birth of affected offspring that escape nature'sscreening mechanism can be averted by the option of prenataldiagnosis. The thrust of prenatal diagnosis is to prevent thetragic impact of debilitating genetic disorders. But notallat-risk parents wish to avail themselves of prenatal diagnosisbecause they are unwilling to accept the choice of therapeuticabortion. Prevention of a genetic disorder before implantationwould obviate the necessity of an abortion at a later stageof pregnancy. With this perspective, the correction of the basicgenetic flaw by replacing the faulty gene with a functioningallele is an attractive alternative. Notwithstanding the imprecisetechnology that presently serves to caution against immediateapplication, gene therapy is a reasonable and natural extensionof efforts to ameliorate the effects of severe inherited disorders.     

The effect of ascorbic acid during biopsy and cryopreservation on viability of bovine embryos produced in vivo

Multiple ovulation embryo transfer (MOET) is used to make more rapid progress in animal breeding schemes. On dairy farms, where female calves are more desired, embryo sex diagnosis is often performed before embryo transfer. Fresh transfers have been favored after biopsy due to cumulative drop in pregnancy rates following cryopreservation. The aim of this study was to explore whether exposure to ascorbic acid (AC) during biopsy and freezing increases the viability of biopsied embryos after cryopreservation. Data on presumptive pregnancy and calving rates of biopsied and cryopreserved/overnight-cultured embryos were gathered. Results showed differences in presumptive pregnancy rates between the groups: 45% for both biopsied-cryopreserved groups (control and AC), 51% for biopsied-overnight-cultured embryos and 80% for intact-fresh embryos. Differences between the groups were also apparent in calving rates: 22% for biopsied-cryopreserved control embryos, 31% for biopsied-cryopreserved AC-embryos, 23% for biopsied-overnight-cultured embryos and 63% for intact-fresh embryos. It is concluded that manipulated embryos are associated with lower presumptive pregnancy and calving rates compared with intact-fresh embryos. The highest calving rates for groups of manipulated embryos were achieved in the AC-group. Therefore, addition of AC can be recommended if biopsy is combined with freezing before transfer.     

Preimplantation genetic diagnosis and embryo research--human developmental biology in clinical practice.

Research on human preimplantation embryos in vitro is controversial. Yet it has been the cornerstone for the development important clinical assisted conception techniques. Preimplantation genetic diagnosis which has developed out of this assisted reproductive technology for the first time provides a realistic alternative to prenatal diagnosis and abortion for couples who are at substantial risk of conceiving a pregnancy affected by a known genetic disorder. It also provides the first real hope of therapy for couples who have suffered repeated miscarriages due to chromosome translocations. However, the ability to test very early embryos in vitro presents new and unusual ethical challenges for clinicians and developmental biologists.     

Successful preimplantation genetic aneuploidy screening in Turkish patients

Preimplantation genetic diagnosis is a preventive approach for identifying genetic abnormalities in early stages of reproduction. We used preimplantation genetic aneuploidy screening in 230 cycles of patients with indications of advanced maternal age, recurrent implantation failure, recurrent spontaneous abortions, or severe male factor. Biopsied blastomeres from embryos with six to eight blastomeres on day 3 were fixed and fluorescence in situ hybridization was utilized on chromosomes 13, 16, 18, 21, 22, X, and Y. Among 945 morphologically normal embryos, 314 were diagnosed as chromosomally normal. Trisomy and monosomy were observed in 36% of the cases (18% each). Embryo transfer was used in 144 cycles, resulting in 41 pregnancies. Thirty-seven healthy babies were delivered, with a take-home baby rate of 24.2% and an implantation rate of 22%. We recommend preimplantation genetic aneuploidy screening as a valuable technique to select normal chromosome embryos in order to avoid multiple pregnancies due to the multiple embryo transfers that are normally necessary to ensure pregnancy in poor prognosis in vitro fertilization patients.     

High rates of embryonic loss, yet high incidence of multiple births in human ART: is this paradoxical?

Humans have low natural fecundity, as the probability of establishing a viable conception in any one menstrual cycle is 20-25% for a healthy, fertile couple. There are numerous underlying causes for this low rate of human fertility, not the least of which are intrinsic abnormalities within the oocyte and/or embryo, which likely account for greater than 50% of failed conceptions. During assisted reproduction technology (ART) interventions, controlled ovarian stimulation is used to obtain several oocytes in attempts to increase the likelihood of having at least one developmentally competent embryo available for transfer. However, current techniques for identifying the competent embryo(s) are by no means perfect. These limitations, coupled with pressures to maximize the chance of pregnancy, typically result in the transfer of multiple embryos. Not surprisingly, this practice has resulted in an unacceptably high rate of multiple pregnancies arising from ART. During the last few years, concerted efforts have focused on reducing these rates. Programs for ART are developing patient-specific policies, restricting the number of embryos to transfer. In addition, strategies are being adopted to improve the accuracy for selecting viable embryos for transfer. One such strategy involves further refinement of morphological criteria associated with improved viability by considering, for example, pronuclei disposition, nucleolar organization, and identification of the fast-cleaving embryos with only mononucleate blastomeres. Another strategy employs pre-implantation genetic diagnosis (PGD) whereby a biopsied blastomere is tested for ploidy using fluorescence in situ hybridization (FISH). A final strategy involves extending the duration of culture to the blastocyst stage, thereby allowing self-selection of those embryos capable of proceeding to blastulation and exclusion of those less viable embryos that succumb to developmental arrest. Together, these strategies are enabling fewer embryos of higher quality to be transferred. Accordingly, the overall pregnancy rate from ART continues to increase, while the rate of triplet and higher order multiple births continues to decline. Nevertheless, the high incidence of intrinsic developmental anomalies in human oocytes inevitably will continue to result in a high degree of embryonic loss in ART.     

Blastomere removal from cleavage-stage mouse embryos alters steroid metabolism during pregnancy

Preimplantation genetic diagnosis (PGD) is a genetic screening of embryos conceived with assisted reproduction technologies (ART). A single blastomere from an early-stage embryo is removed and molecular analyses follow to identify embryos carrying genetic defects. PGD is considered highly successful for detecting genetic anomalies, but the effects of blastomere biopsy on fetal development are understudied. We aimed to determine whether single blastomere removal affects steroid homeostasis in the maternal-placental-fetal unit during mouse pregnancy. Embryos generated by in vitro fertilization (IVF) were biopsied at the four-cell stage, cultured to morula/early blastocyst, and transplanted into the oviducts of surrogate mothers. Nonbiopsied embryos from the same IVF cohorts served as controls. Cesarean section was performed at term, and maternal and fetal tissues were collected. Embryo biopsy affected the levels of steroids (estradiol, estrone, and progesterone) in fetal and placental compartments but not in maternal tissues. Steroidogenic enzyme activities (3beta-hydroxysteroid dehydrogenase, cytochrome P450 17alpha-hydroxylase, and cytochrome P450 19) were unaffected but decreased activities of steroid clearance enzymes (uridine diphosphate-glucuronosyltransferase and sulfotransferase) were observed in placentas and fetal livers. Although maternal body, ovarian, and placental weights did not differ, the weights of fetuses derived from biopsied embryos were lower than those of their nonbiopsied counterparts. The data demonstrate that blastomere biopsy deregulates steroid metabolism during pregnancy. This may have profound effects on several aspects of fetal development, of which low birth weight is only one. If a similar phenomenon occurs in humans, it may explain low birth weights associated with PGD/ART and provide a plausible target for improving PGD outcomes.     

Birth of normal infants after transfer of embryos that were twice vitrified/warmed at cleavage stages: Report of two cases

The role of cryopreservation in assisted reproductive technology programs has increased within the last years allowing the transfer of a limited number of embryos and the storage of the remaining for future use. The reduction in the number of transferred embryos decreases the frequency of multiple pregnancy rates and of ovarian hyperstimulation syndrome while the cumulative pregnancy rate can be maximized. Moreover, as not all embryos will survive the warming process more cleavage stage embryos are warmed to improve selection for transfer. Therefore, surplus good quality cleavage stage embryos and/or blastocysts must be re-vitrified for further transfer to achieve pregnancy. To our knowledge, there have been no reports demonstrating that human embryos can be successfully vitrified/warmed twice at the cleavage stage. Thus we report two successful pregnancies and deliveries of healthy babies after transfer of embryos that were twice vitrified/warmed at 2–4 cells stage.     

Birth of normal infants after transfer of embryos that were twice vitrified/warmed at cleavage stages: Report of two cases

The role of cryopreservation in assisted reproductive technology programs has increased within the last years allowing the transfer of a limited number of embryos and the storage of the remaining for future use. The reduction in the number of transferred embryos decreases the frequency of multiple pregnancy rates and of ovarian hyperstimulation syndrome while the cumulative pregnancy rate can be maximized. Moreover, as not all embryos will survive the warming process more cleavage stage embryos are warmed to improve selection for transfer. Therefore, surplus good quality cleavage stage embryos and/or blastocysts must be re-vitrified for further transfer to achieve pregnancy. To our knowledge, there have been no reports demonstrating that human embryos can be successfully vitrified/warmed twice at the cleavage stage. Thus we report two successful pregnancies and deliveries of healthy babies after transfer of embryos that were twice vitrified/warmed at 2–4 cells stage.     

植入前遗传学诊断的原理、方法及适应症

植入前遗传学诊断是一种非常早的产前诊断,指在胚胎着床之前即对配子或胚胎的遗传物质进行分析,检测配子或胚胎是否有遗传物质异常,选择正常胚胎进行移植。与传统的产前诊断相比,能避免选择性流产异常妊娠给妇女带来的心身痛苦。本文就该领域的发展及现状和其诊断原理、方法及适应症进行了总结和综述。 Abstract:Preimplatation genetic diagnosis (PGD) is a very early form of prenatal diagnosis.Gametes or embryos are biopsied and a genetic diagnosis is carried out on the biopsied cells to investigate if the gametes or embryos is free of genetic disease.And the normal embryos is transferred to the mother.Comparing to the traditional prenatal diagnosis,PGD is a method that can avoid aborting a abnormal pregnant and reduce pains of women.In this review,we introduce the history of development and statues in quo,principle,method and application of PGD.     

Molecular cytogenetic studies towards the full karyotype analysis of human blastocysts and cytotrophoblasts

Numerical chromosome aberrations in gametes typically lead to failed fertilization, spontaneous abortion or a chromosomally abnormal fetus. By means of preimplantation genetic diagnosis (PGD), we now can screen human embryos in vitro for aneuploidy before transferring the embryos to the uterus. PGD allows us to select unaffected embryos for transfer and increases the implantation rate in in vitro fertilization programs. Molecular cytogenetic analyses using multi-color fluorescence in situ hybridization (FISH) of blastomeres have become the major tool for preimplantation genetic screening of aneuploidy. However, current FISH technology can test for only a small number of chromosome abnormalities and hitherto failed to increase the pregnancy rates as expected. We are in the process of developing multi-color FISH-based technologies to score all 24 chromosomes in single cells within a three-day time limit, which we believe is vital to the clinical setting. Also, human placental cytotrophoblasts (CTBs) at the fetal-maternal interface acquire aneuploidies as they differentiate to an invasive phenotype. About 20-50% of invasive CTB cells from uncomplicated pregnancies were found to be aneuploid, suggesting that the acquisition of aneuploidy is an important component of normal placentation, perhaps limiting the proliferative and invasive potential of CTBs. Since most invasive CTBs are interphase cells and possess extreme heterogeneity, we applied multi-color FISH and repeated hybridizations to investigate the feasibility of a full karyotype analysis of individual CTBs. In summary, this study demonstrates the strength of Spectral Imaging analysis and repeated hybridizations, which provides a basis for full karyotype analysis of single interphase cells.     

Preimplantation genetic diagnosis--an overview.

Since the early 1990s, preimplantation genetic diagnosis (PGD) has been expanding in scope and applications. Selection of female embryos to avoid X-linked disease was carried out first by polymerase chain reaction, then by fluorescence in situ hybridization (FISH), and an ever-increasing number of tests for monogenic diseases have been developed. Couples with chromosome rearrangements such as Robertsonian and reciprocal translocations form a large referral group for most PGD centers and present a special challenge, due to the large number of genetically unbalanced embryos generated by meiotic segregation. Early protocols used blastomeres biopsied from cleavage-stage embryos; testing of first and second polar bodies is now a routine alternative, and blastocyst biopsy can also be used. More recently, the technology has been harnessed to provide PGD-AS, or aneuploidy screening. FISH probes specific for chromosomes commonly found to be aneuploid in early pregnancy loss are used to test blastomeres for aneuploidy, with the aim of replacing euploid embryos and increasing pregnancy rates in groups of women who have poor IVF success rates. More recent application of PGD to areas such as HLA typing and social sex selection have stoked public controversy and concern, while provoking interesting ethical debates and keeping PGD firmly in the public eye.     

Detection of structural and numerical chromosome abnormalities in interphase cells using spectral imaging.

Chromosome abnormalities are common causes of congenital malformations and spontaneous abortions. They include structural abnormalities, polyploidy, trisomy, and mosaicism. In in vitro fertilization (IVF) programs, preimplantation genetic diagnosis (PGD) of oocytes and embryos has become the technique of choice to select against abnormal embryos before embryo transfer. For diagnosis of structural abnormalities, we developed case-specific breakpoint-spanning DNA probes. Screening of an in-house yeast artificial chromosome (YAC) library is facilitated by information from publicly available databases and published articles. Most numerical chromosome abnormalities, on the other hand, are detrimental to early embryonic development and increase with maternal age. We therefore developed a multichromosome screening technique based on spectral imaging to simultaneously detect and score as many as 10 different chromosome types. The probe set was chosen to detect more than 70% of all numerical chromosome aberrations responsible for spontaneous abortions. Detecting structural and numerical abnormalities in single interphase cells using spectral imaging is a powerful technique for multilocus genetic screening.     

Preimplantation genetic diagnosis: the German situation

Preimplantation genetic diagnosis makes it possible to detect some genetic disorders in embryos in vitro before they are transferred to the uterus. Using this technique, there is an opportunity for couples who have an increased risk of transmitting severe genetic disorders to their offspring to reduce this risk by >95%. By doing PGD, abortions at a later stage can be avoided.     

The genetic screening of preimplantation embryos by comparative genomic hybridisation

Comparative genomic hybridization (CGH) is an indirect DNA-based test which allows for the accurate analysis of aneuploidy involving any of the 24 types of chromosomes present (22 autosomes and the X and Y sex chromosomes). Traditionally, embryos have been screened using fluorescence in situ hybridization (FISH)--a technique that was limited in the number of chromosomes able to be identified in any one sample. Early CGH reports on aneuploidy in preimplantation embryos showed that any of the 24 chromosomes could be involved and so FISH methods were going to be ineffective in screening out abnormal embryos. Our results from routine clinical application of array CGH in preimplantation genetic diagnosis (PGD) patients confirm previous reports on patterns of chromosomal contribution to aneuploidy. The pregnancy outcomes following embryo transfer also indicate that despite the requirement to freeze embryos, rates are encouraging, and successful ongoing pregnancies can be achieved.     

Influence of inbreeding on reproductive performance, ejaculate quality and testicular volume in the dog

The influence of fast freezing and thawing on bovine embryos at different stages of development was investigated. A total of 20 day-7 embryos and 37 day-8 embryos were thawed and classified morphologically before being transferred nonsurgically to synchronized recipients. Ninety percent (18 20 ) of the day 7 embryos (late morulae and blastocysts) were classified as transferable and a pregnancy rate of 52.9% (9 17 ) was obtained with these embryos. Seventy eight percent (29 37 ) of the day 8 embryos (expanded blastocysts) were classified as transferable, but only 24.1% (7 29 ) of these embryos resulted in pregnancy. The best pregnancy rate was obtained with the blastocysts of day 7 (5 8 or 62.5%), which compares favorably with that of freshly collected embryos. It is suggested that the low pregnancy rate found in the day 8 embryos is related to ultrastructural damages of the desmosomes and tonofilaments within the trophoblast layer, which results in a disturbance of the normal hatching process.     

Preimplantation genetic diagnosis of β-thalassemia using real-time polymerase chain reaction with fluorescence resonance energy transfer hybridization probes

Preimplantation genetic diagnosis (PGD) is employed increasingly to allow transfer of embryos to the uterus in assisted reproduction procedures. There are three stages of biopsy: polar bodies, one or two blastomeres from the cleavage-stage embryos, and trophectoderm cells (∼5 cells) from the blastocyst-stage embryos. Validation of polymerase chain reaction (PCR)-based assays are challenging because only limited genetic material can be obtained for PGD. In the current study, we modified a valid single-cell PCR protocol for PGD using real-time PCR assay with fluorescence resonance energy transfer (FRET) hybridization probes followed by melting curve analysis. We optimized and clinically applied the protocol, permitting molecular genetic analysis to amplify a specific region on the beta-globin (HBB) gene for a couple, carriers of two mutations: c.-78A>G and c.52A>T. Among a total of eight embryos obtained after ovarian stimulation, a single blastomere per embryo at the six- to eight-cell stage was biopsied. This PGD method showed that four embryos were unaffected, two embryos were selected for transfer, and one pregnancy was achieved. Finally, a healthy male baby was delivered at 38 weeks’ gestation. The results obtained using the new method, FRET hybridization probes, were compared with findings using an existing method, primer extension minisequencing.