Efficacy and clinical outcome of preimplantation genetic diagnosis using FISH for couples of reciprocal and Robertsonian translocations: the Korean experience

OBJECTIVE: To evaluate the efficacy and clinical outcome of preimplantation genetic diagnosis (PGD) using fluorescence in situ hybridization (FISH) for couples with chromosomal translocations. METHODS: PGD using FISH was performed in 59 cycles of 43 couples with reciprocal translocations, and 11 cycles of 6 couples with Robertsonian translocations. The diagnostic and clinical data were reviewed in a series of 70 treatment cycles of 49 couples from January 2001 to June 2002 at Samsung Cheil Hospital, Korea. RESULTS: A total of 1408 oocytes were retrieved, and 938 (81.7%) out of 1148 matured oocytes were fertilized by intracytoplasmic sperm injection (ICSI). Single blastomere biopsy and FISH analysis were successfully carried out in 99.3% (890/896) and 94.4% (840/890), respectively. Among 193 normal or balanced embryos, 169 embryos were transferred in 64 cycles (91.4% per started cycle). Twenty clinical pregnancies including two ectopic pregnancies and three spontaneous miscarriages (28.6% per started cycle, 31.3% per transfer cycle, 40.8% per couple) were established. Of the three spontaneous miscarriages, one was karyotyped as normal, one had an unbalanced arrangement and one was tetraploid. One case of preterm twin delivery occurred and 16 healthy babies were delivered in 12 single and 2 twin pregnancies. CONCLUSION: The clinical outcome was successful in 28.6% (14/49) of the treated couples with translocations after PGD. The spontaneous abortion rate was significantly reduced from 95.8% (69/72) to 16.7% (3/18) in these couples.     

Preimplantation genetic diagnosis by fluorescence in situ hybridization of reciprocal and Robertsonian translocations

ObjectiveThe presence of reciprocal and Robertsonian chromosomal rearrangement is often related to recurrent miscarriage. Using preimplantation genetic diagnosis, the abortion rate can be decreased. Cases treated at our center were reviewed.Materials and methodsA retrospective analysis for either Robertsonian or reciprocal translocations was performed on all completed cycles of preimplantation genetic diagnosis at our center since the first reported case in 2004 until the end of 2010. Day 3 embryo biopsies were carried out, and the biopsied cell was checked by fluorescent in situ hybridization using relevant informative probes. Embryos with a normal or balanced translocation karyotype were transferred on Day 4.ResultsThirty-eight preimplantation genetic diagnosis cycles involving 17 couples were completed. A total of 450 (82.6%) of the total oocytes were MII oocytes, and 158 (60.0%) of the two-pronuclei embryos were biopsied. In 41.4% of the fluorescent in situ hybridization analyses, the results were either normal or balanced. Embryos were transferred back after 21 cycles. Three babies were born from Robertsonian translocation carriers and another two from reciprocal translocation carriers. The miscarriage rate was 0%. Among the reciprocal translocation group, the live delivery rate was 8.3% per ovum pick-up cycle and 18.2% per embryo transfer cycle. Among the Robertsonian translocation group, the live delivery rate was 14.3% per ovum pick-up cycle and 20.0% per embryo transfer cycle.ConclusionThere is a trend whereby the outcome for Robertsonian translocation group carriers is better than that for reciprocal translocation group carriers. Aneuploidy screening may possibly be added in order to improve the outcome, especially for individuals with an advanced maternal age. The emergence of an array-based technology should help improve this type of analysis.     

Embryo development characteristics in Robertsonian and reciprocal translocations: a comparison of results with non-translocation cases

The effect of translocations on embryo development was evaluated and results were compared in terms of embryo development with those of embryos obtained from standard intracytoplasmic sperm injection (ICSI) cycles. In 23 translocation carriers with 34 cycles, fertilization, pronuclear morphology scoring (PMS), developmental arrest, cleavage and blastocyst formation were evaluated and compared with embryos obtained from non-translocation cases undergoing ICSI (n = 98 cycles). In 28 cycles, preimplantation genetic diagnosis (PGD) was performed on prezygotes (first and second polar body biopsy for female carriers; n = 3) or on embryos having seven or more blastomeres (blastomere biopsy; n = 25). In six cycles for four couples, probes for translocated chromosomes were not available, so PGD could not be performed. Overall, in translocation cases, a lower fertilization rate, a higher rate of retarded embryo development, and a lower rate of blastocyst formation were observed compared with embryos of non-translocation cases. Fluorescence in-situ hybridization (FISH) analysis showed a 70.9% abnormality rate for reciprocal translocations and 55.0% for Robertsonian translocations respectively. In cases with Robertsonian and reciprocal translocation carriers, the probability of poor embryo development, which may be a result of high segregation abnormalities, may negatively affect the outcome of assisted reproductive techniques. This poor prognosis should also be considered when genetic counselling for translocation is given.     

Clinical application of next-generation sequencing in preimplantation genetic diagnosis cycles for Robertsonian and reciprocal translocations

Purpose

The purpose of this study was to apply next-generation sequencing (NGS) technology to identify chromosomally normal embryos for transfer in preimplantation genetic diagnosis (PGD) cycles for translocations.

Methods

A total of 21 translocation couples with a history of infertility and repeated miscarriage presented at our PGD clinic for 24-chromosome embryo testing using copy number variation sequencing (CNV-Seq).

Results

Testing of 98 embryo samples identified 68 aneuploid (69.4 %) and 30 (30.6 %) euploid embryos. Among the aneuploid embryos, the most common abnormalities were segmental translocation imbalances, followed by whole autosomal trisomies and monosomies, segmental imbalances of non-translocation chromosomes, and mosaicism. In all unbalanced embryos resulting from reciprocal translocations, CNV-Seq precisely identified both segmental imbalances, extending from the predicted breakpoints to the chromosome termini. From the 21 PGD cycles, eight patients had all abnormal embryos and 13 patients had at least one normal/balanced and euploid embryo available for transfer. In nine intrauterine transfer cycles, seven healthy babies have been born. In four of the seven children tested at 18 weeks gestation, the karyotypes matched with the original PGD results.

Conclusion

In clinical PGD translocation cycles, CNV-Seq displayed the hallmarks of a comprehensive diagnostic technology for high-resolution 24-chromosome testing of embryos, capable of identifying true euploid embryos for transfer.

     

The number of eight-cell embryos is a key determinant for selecting day 3 or day 5 transfer

OBJECTIVE: To select patients for day 3 vs. day 5 embryo transfer. DESIGN: Retrospective analysis of assisted reproduction technology (ART) cycles comparing outcomes of day 3 and day 5 transfers. SETTING: ART program of Brigham and Women's Hospital. PATIENT(s): Patients with day 3 or day 5 embryo transfers (n = 221 and 141, respectively). INTERVENTION(s): Cycles with eight or more zygotes were stratified by the number of eight-cell embryos available on day 3 (none, one or two, or three or more). MAIN OUTCOME MEASURE(s): Number of blastocysts, implantation rates, ongoing pregnancy rates, and number of fetal heart beats. RESULT(s): With no eight-cell embryos on day 3, 0% and 33% pregnancies resulted from day 5 vs. day 3 transfers. With one or two eight-cell embryos on day 3, ongoing and high order multiple rates were not different between day 3 and day 5 transfers. With three or more eight-cell embryos, day 5 transfer resulted in a decrease in multiple gestations but no difference in ongoing pregnancy rates compared with day 3 transfer. CONCLUSION(s): With no eight-cell embryos on day 3, a day 3 transfer is warranted. With one or two eight-cell embryos, any benefit of day 5 transfer appears to be equivocal. With three or more eight-cell embryos, day 5 transfer is recommended.     

Clinical outcome of day 2 versus day 5 transfer in cycles with one or two developed embryos

OBJECTIVE: To determine whether extended culture of embryos to blastocysts has any benefit in cycles with only one or two created embryos. DESIGN: Retrospective analysis of cycles comparing outcomes of day 2 and day 5 transfers. Our day 2 group was from the year 1999 and our day 5 group, from the year 2000. SETTING: Assisted reproductive technology program of a teaching hospital. PATIENT(S): All patients, irrespective of age, who had developed one or two embryos. INTERVENTION(S): Stimulated IVF, intracytoplasmic sperm injection, or testicular sperm extraction and intracytoplasmic sperm injection cycles with 2-day culture in universal IVF medium (n = 133) or 5-day culture in BlastAssist media (MediCult, Jyllinge, Denmark; n = 132). MAIN OUTCOME MEASURE(S): Pregnancy, implantation, and take-home baby rates. RESULT(S): In the groups of 2-day and 5-day culture, embryo transfer was performed in 98% and in 57% of cycles, respectively. However, the total implantation rate per created embryo (18% vs. 18%), the pregnancy rate per cycle (23% vs. 21%), and the take-home baby rate (69.4% vs. 71.4%) did not differ between the day 2 and day 5 groups. CONCLUSION(S): Extended culture of embryos does not improve or decrease their capacity for implantation but only allows for better selection and is therefore not necessary in cycles with fewer than three embryos.     

Thawing day 3 embryos and culturing to day 5 may be a better method for frozen embryo transfer

Purpose

Does thawing cleavage embryos and culturing them for transfer as blastocysts improve pregnancy and perinatal outcomes compared to transferring thawed blastocysts?

Methods

Retrospective, observational cohort study performed at two assisted reproductive technology centers, 2014 to 2020. A total of 450 patients with 463 thawed embryo transfer cycles were divided into 2 groups according to the embryonic developmental stage at cryopreservation and transfer: 231 thawed blastocysts (day 5 group) and 232 thawed cleavage embryos that were cultured for 2 days and transferred as blastocysts (day 3–5 group). The two groups were compared for demographics, routine parameters of IVF treatment, pregnancy rates, and perinatal outcomes.

Results

Multivariable logistic regression analysis for ongoing pregnancy and delivery demonstrated that the day 3–5 group had a greater likelihood of achieving ongoing pregnancy and delivery compared to the day 5 group (OR 1.58, 95%CI 1.062–2.361, p?=?0.024). Perinatal outcomes were comparable between the three groups.

Conclusion

Our results support culturing post-thaw cleavage embryos for 2 days and transferring them as blastocysts to increase chances of ongoing pregnancy and delivery.

     

Relationship between peak serum estradiol levels and treatment outcome in in vitro fertilization cycles after embryo transfer on day 3 or day 5

OBJECTIVE: To examine the relationships between peak serum estradiol (E(2)) levels and treatment outcome in in vitro fertilization (IVF) cycles after embryo transfer (ET) on day 3 or day 5. DESIGN: Retrospective analysis of 697 IVF-ET cycles between January 1999 and December 2001. SETTING: A university-affiliated assisted reproduction program. PATIENT(S): Infertile patients undergoing IVF-ET cycles. INTERVENTION(S): Peak E(2) concentration in serum was determined on the day of human chorionic gonadotropin (hCG) administration. The IVF-generated embryos were cultured for 2 days until transfer on day 3. If more than four 8-cell embryos were present on day 3, embryo culture was continued until day 5 for blastocyst transfer. MAIN OUTCOME MEASURE(S): Clinical pregnancy rates. RESULT(S): High peak E(2) levels did not adversely affect treatment outcome. After the cycles were divided according to the day of ET, high peak E(2) levels were associated with improved pregnancy rates after ET on day 5 but not on day 3. CONCLUSION(S): Increasing peak E(2) levels in IVF cycles are associated with improved pregnancy rates after ET on day 5.     

Comparison of aneuploidy,pregnancy and live birth rates between day 5 and day 6 blastocysts

Comprehensive chromosome screening is typically used for aneuploidy analysis of blastocysts. It is believed that either day of blastocyst development is acceptable. Euploidy rates and outcomes were examined between day 5 and day 6 blastocysts in two studies. First, euploidy rates of day 5 and day 6 blastocysts were examined on a per-embryo and per-patient basis. Second, outcomes were compared when only euploid day 5 or day 6 blastocysts were transferred in a cryopreserved embryo transfer cycle. In cycles (n = 70) that had blastocysts biopsied on both day 5 and day 6, day 5 blastocysts had a higher chance of being euploid than day 6 blastocysts (125/229 [54.6%]) and (77/180 [42.8%]), respectively (P = 0.0231). Similarly, euploid rates in blastocysts from patients (n = 193) with day 5 biopsy, day 6 biopsy, or both, were significantly higher in day 5 (235/421 [55.8%]) compared with day 6 (184/413 [44.6%]) blastocysts (P = 0.0014). In the second study, 50 women (36.1 ± 4.3 years) and 39 women (35.1 ± 3.8 years) with only euploid day 5 or euploid day 6 blastocysts transferred during a cryopreserved embryo transfer had similar cycle outcomes. Although underpowered, these data suggest that euploid day 6 blastocysts are as capable of positive outcomes as their euploid day 5 counterparts.     

Evaluation of 1100 couples with recurrent pregnancy loss using conventional cytogenetic,PGD, and PGS: hype or hope

Recurrent pregnancy loss (RPL) is an important clinical problem, mostly resulting from chromosomal or genetic defects, while in 30–60% of cases, it is idiopathic. The aim of this study is to evaluate the frequency and types of chromosomal abnormalities, also pre-implantation genetic diagnosis (PGD) and pre-implantation genetic screening (PGS) outcomes among Iranian couples with RPL. This retrospective study was conducted on 1100 Iranian couples (2200 individuals) with RPL referred to Royan Institute between 2008 and 2014. Karyotyping had been performed using standard cytogenetic techniques. PGD results of RPL patients with abnormal karyotypes and PGS results of RPL patients with normal karyotypes were also analyzed. The frequency of chromosomal abnormalities in these patients was 4.95%. Women demonstrated more abnormalities (6.82%) in comparison to men (3.09%). The successful rate of pregnancy after PGD and PGS was 52 and 18.64%, respectively. The observation of 4.95% chromosomal abnormalities among the patients with RPL could support this hypothesis that there is a direct relationship between chromosomal abnormalities and RPL. More than half of the patients who underwent PGD had successful pregnancy; therefore, this approach can improve the success rate of pregnancy in them. The results of PGS cycles showed that this technique could increase the live birth rate in RPL patients.     

Analysis of meiotic segregation patterns and interchromosomal effects in sperm from six males with Robertsonian translocations

Purpose To provide more genetic information about meiotic segregation behavior and the possibility of interchromosomal effects (ICE) in spermatozoa from carriers of Robertsonian (Rob) translocations. Materials and methods Meiotic segregation behavior in spermatozoa from six carriers of Rob translocations, four t(13;14), one t(14;22) and one t(13;21), was investigated by dual fluorescence in-situ hybridization (FISH). Aneuploidy for chromosomes 18, X and Y was studied by triple FISH. Results The rate of normal/balanced spermatozoa resulting from alternate segregation ranged from 78.14 to 86.88%. The frequency of unbalanced spermatozoa resulting from adjacent segregation varied between 11.70 and 19.53%. The higher frequencies of aneuploidy for sex chromosome were observed in three Rob translocation carriers. In addition, the increased rates of diploid were found in two t(13;14) carriers. Conclusions Alternate segregation is dominant in the different types of Rob translocations. Some carriers may be at an increased risk for ICE. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. An increased aneuploidy for sex chromosome observed in three Robertsonian translocation carriers suggests that an interchromosomal effect is likely in some carriers.     

Establishment of criteria for elective single embryo transfer at day 2 or day 3 by analyzing cases with successful implantation of all embryos transferred

AIM: Elective transfer of two good-quality embryos has been used to avoid triplet or high-order multiple pregnancies. However, the rate of twin pregnancies has remained fairly unchanged. In the present study, criteria for elective single embryo transfer (eSET) at day 2 or day 3 were established by analyzing cases with successful implantation of all embryos transferred. METHODS: A total of 685 fresh or frozen-thawed embryo transfers following in vitro fertilization/intracytoplasmic sperm injection between April 2002 and March 2006 were performed. Only embryo transfers at day 2 or day 3, but not at blastocyst stage, were included. Successful implantation of all embryos transferred was obtained in 17 pregnancy cycles. RESULTS: Thirty-one gestational sacs with fetal heartbeats were obtained by a total of 31 embryo transfers in 17 infertile women. The average age was 32.6 years (23-38), and 14 (82.3%) of the 17 women were <36 years old. Fifteen (88.2%) of the 17 pregnancies were established at the first attempt of assisted reproductive technology (ART). Of the 17 women, eight (47.1%) women were multigravida and four (23.5%) women were multipara. The indications for ART or insemination methods did not seem to be related to the pregnancy results. Twenty-nine (93.5%) of 31 embryos implanted were considered good-quality embryos. Of the 17 fresh embryos transferred at day 2, 15 were at the 4-cell stage and two were at the 5-cell stage. Of the 11 fresh embryos transferred at day 3, one was at the 6-cell stage, two were at the 7-cell stage and eight were at the 8-cell stage. CONCLUSION: The criteria for eSET at day 2 or day 3 were established as follows: <36 years of age, a first treatment cycle and more than two good-quality embryos developed at least to the 4-cell stage at day 2, or 6-cell stage at day 3. Additionally, the past history of pregnancy or delivery should be considered, as patients positive for such history might have better implantation ability. eSET can be highly recommended to avoid twin pregnancies in subjects with the established criteria.