Blastocyst Stage Biopsy Research Articles

O-322 What is the number of IVF cycles needed to produce at least one euploid blastocyst in patients of all ages with varied AMH levels

Abstract Study question How many cycles are needed to get at least one blastocyst in patients of different AMH levels and varied ages? Summary answer Age and AMH determine the number of cycles, which ranges from 1 cycle in young patients up to 10 cycles in over-44-year-old patients. What is known already AMH secretion by ovarian granulosa cells initiates during the 36th week of gestation in pre-antral and antral follicles. This persists throughout a woman’s reproductive lifespan. AMH serves as a biomarker for assessing ovarian reserve and forecasting the ovarian response to stimulation. AMH is strongly associated with the quantity of antral follicles and the production of oocytes. AMH can be utilised to determine the initial dosage of FSH in an IVF cycle, as well as serving as a quantitative indicator of the number of oocytes produced. Study design, size, duration In this retrospective cohort study, data was collected from 16,417 ICSI cycles with Preimplantation Testing for Aneuploidy (PGT-A) conducted at three referral fertility centers from Jan 2019 to Dec 2023. We excluded cycles without serum AMH, endocrinopathies or recurrent pregnancy loss, males with total sperm count of less than 5 million and total motility less than 30%, and those with parental chromosomal abnormalities. Participants/materials, setting, methods Patients were stratified into three different categories of AMH (ng/ml), low (< 1.1 ng/mL), intermediate (>1.1 to < 3.0 ng/mL) and high (> 3.0 ng/mL). Each category was subdivided into 6 age sub-categories as follow (< 35), (35 to < 38), (38 to < 40), (40 to < 42), (42 to < 44), (> 44). All of the cycles underwent blastocyst stage biopsy and PGT-A utilising next generation sequencing (NGS). Main results and the role of chance Euploidy rate was statistically insignificant for all AMH levels in the same age group. However, euploid embryo numbers vary significantly. Euploid embryos numbers are shown in low, medium, and high AMH by age group. In the (<35) years was 1.9±1.5, 2.9±2.3, and 4.7±3.5, p < 0.0001. In the (35 to > 38) years was 1.3±0.8, 2.1±1.8, and 3.2±2.6, p < 0.0001. In the (38 to > 40) years was 0.9±0.8, 1.7±1.4, and 2.6±1.7, p < 0.0001. In the (40 to > 42) years was 0.6±0.5, 0.9±0.7, and 1.5±1.4, p < 0.0001. In the (42 to > 44) year was 0.26±0.2, 0.49±0.38, and 0.6±53, p < 0.0001. In the (≥44) years was 0.10±0.10, 0.23±0.21, and 0.40±0.37, p = 0.0170. We calculated the number of cycles needed to produce at least one grade BB or higher euploid blastocyst from the mean number of euploid embryos per cycle: The number of cycles is presented in low, medium, and high AMH. In the (<35) and (35 to > 38), 1 cycle is needed. In (38 to > 40) years, 2 cycles, 1 cycle and 1 cycle. In the (40-42) group 2 cycles, 2 cycles and 1 cycle. In the (42 to < 44) group 4 cycles, 3 cycles and 2 cycles. In the (≥44) group 10 cycles, 5 cycles and 3 cycles. Limitations, reasons for caution The retrospective nature of the study is the main limitation. Wider implications of the findings The study offers valuable insights for patient counselling regarding the required number of cycles to achieve euploid embryos, as well as for financial budgeting for both patients and insurance companies. Trial registration number Not applicable

Preimplantation genetic testing for aneuploidies (abnormal number of chromosomes) in in vitro fertilisation.

Preimplantation genetic testing for aneuploidies (abnormal number of chromosomes) in in vitro fertilisation.

PROGRESS TOWARDS UNIVERSAL PGT- HOW CLOSE ARE WE TO COMPREHENSIVE PGT?

Are we there yet?- these are the words that many parents would have heard when taking children on some sort of trip. The journey began over 30 years ago when the first PGTs were done. It was simple back then- the answers required were generally simple (or at least straightforward) and you only went looking for directed answers. PGS was for chromosomes and PGD was for a gene(s)- and generally, never the two would meet. Early chromosome testing was based on FISH and the targets were polar bodies or blastomeres from cleavage embryos- apart from some technical expertise in getting the sample, analysis was essentially down to the operator's ability to count to 1 (2nd PB) or 2 (1st PB and blastomeres). Life was good- but unfortunately the results were often not so productive with implantation rates resisting change and only some groups reporting decreased miscarriage rates for some patient cohorts. The naysayers reached a crescendo around 2005 when it was proclaimed that not only was PGS not helping but it was hurting patient's chances of a pregnancy. Three years of gloom followed with claim and counter claim on the appropriateness of PGS. PGD fared a bit better with the simple logic of Mendelian genetics and obvious success in outcomes- although some mishaps in the early days led some groups to possibly over emphasize and focus on DNA contamination, ADO and recombination events- life for PGD though was generally good. The darkness of those 3 years of doubt and dispute were substantially dispelled when a British Knight led the field into the new era of comprehensive chromosome screening where every chromosome could now be seen to be both problematic and mischievous. Finally, the full impact of identifying any and all chromosome changes was seen- a dramatic increase in implantation rate and decreased miscarriage rates for most groups tested. There were still some shadowy areas of understanding but the light was again shining and things were, maybe, good again. Was it just a breakthrough in analysis that flicked the switch? No, there were some underlying bits to the puzzle that were not given the amount of the credit for the improvements that they possibly deserved. Two other steps in addition to CCS were essential for the realization of these new outcomes- vitrification with concomitant cryo-transfer and blastocyst stage biopsy. The naysayers retreated, not totally but sufficiently far to encourage the bulk of the world to adopt the new processes. Are we there yet?. No, but getting closer. In the meantime, PGD continued on its singular path but now it had to also face the multitude of positive findings associated with the benefits of CCS- find not only the correct genetics for an embryo but also the correct chromosome compliment. How to combine the two for the best of both world's outcomes? Approaches to improved PGS (now referred to as PGT-A, PGT-SR) and combining PGD (now PGT-M) into a single will be discussed. Are we there yet?

Preimplantation genetic testing for aneuploidy (PGT‐A): The biology, the technology and the clinical outcomes

Preimplantation genetic testing for aneuploidy (PGT-A) seeks to identify preimplantation embryos with a normal chromosome complement (euploid) during in vitro fertilisation (IVF). By sifting out embryos with abnormal chromosome numbers (aneuploid), PGT-A should theoretically improve pregnancy success. However, earlier versions of PGT-A were ineffective, and in some cases, detrimental, due to biopsy-induced trauma and because the technology at the time could analyse only a fraction of all chromosomes. More recently, the emergence of technologies enabling all chromosomes to be analysed and a switch to less traumatic blastocyst-stage biopsy have seen widespread uptake of PGT-A. Assessing the full impact of blastocyst biopsy PGT-A requires consideration of multiple factors, including embryonic mosaicism, sensitivity of the technological platform used, embryo loss during long-term in vitro culture, embryo cryopreservation and inter-clinic variability in expertise. Significantly, there hasnt yet been an appropriately designed randomised controlled trial (RCT) of blastocyst biopsy PGT-A analysed by intention-to-treat that accounts for all these parameters on a per-cycle basis. The three RCTs reporting benefits studied outcomes on a per-embryo transfer basis were small and underpowered and demonstrated benefits for a very select sub-group of good prognosis patients. The liberal use of this very expensive IVF add-on for other patient populations has not yet been shown to be effective, or indeed, without harm.

Assessment of Chromosomal Aneuploidy in Day 3 Cleavage-Stage Embryos and Day 5/6 Blastocysts from Indigenous Black Africans

The objectives of this study were to examine the prevalence and to characterize the types of chromosomal aberrations of aneuploidy in cleavage-stage Day 3 human embryos and in Day 5/6 human blastocysts in indigenous Black African women aged ≤ 35 and >35 years. Of the 312 oocytes fertilized for Day 3 embryos and 269 for Day 5/6 blastocysts, 230 blastomeres and 122 blastocysts were biopsied respectively and then subjected to genetic analysis using the array CGH and next generation sequencing methods, genotyping to characterize chromosomal error types of aneuploidy across all 24 chromosomes. A total of 562 oocytes with 486 MII oocytes, were fertilized from which 230 Day 3 embryos and 122 Day 5/6 blastocysts were biopsied. Euploidy rates in Day 3 embryo group and in Day 5/6 blastocyst group were 24.3% and 75.0% respectively while aneuploidy rates were 75.7% and 25.0% respectively. Women aged ≤ 35 years were approximately thrice as likely to have euploid Day 3 embryos (Fisher’s χ²=1.18, p-value=0.29, OR=2.79, 95% CI: 0.61, 12.67). Blastocyst stage biopsy was more efficient in PGS. Blastocyst culture had some level of aneuploidy selection. Higher diagnosis rate was observed as there was lower rate of embryos with no diagnosis.

Clinical analysis of preimplantation genetic diagnosis with HLA matching for beta-thalassemia

To investigate the efficacy and feasibility of preimplantation genetic diagnosis(PGD)with human leukocyte antigen(HLA)matching for beta-thalassemia. A total of 43 referred beta-thalassemia couples, with at least on child in need of hematopoietic stem cell transplantation(HSCT), underwent PGD for HLA matching at the First Affiliated Hospital of Sun Yat-sen University from 2010 to 2015. PGD cycles of these couples were retrospectively analyzed, and 15 infants born from PGD-HLA were followed up. A total of 84 oocyte retrieval cycles were performed, providing 14±7 oocytes per cycle. Fifty nine embryos biopsied cycles were done, including 24 cleavage stage and 35 blastocyst stage biopsy cycles. In cleavage stage, 259 embryos were biopsied, 93.4%(242/259)of them with conclusive molecular diagnosis, and the percentage of unaffected embryos(normo-homozygote and heterozygote)was 71.4%(185/259). The percentage of HLA matched unaffected embryos was 9.3%(24/259). In blastocyst stage, 306 embryos were biopsied, while 93.8%(287/306)of them were conclusive, and the percentage of unaffected embryos was 70.6%(216/306). The percentage of HLA matched unaffected embryos in blastocyst stage biopsy was 14.4%(44/306), which was higher than in cleavage stage biopsy(P< 0.05). Forty three female carriers underwent 48 embryo transfer cycles including 3 fresh and 45 frozen-thawed embryo transfer cycles. Three fresh embryo transfer cycles were done after cleavage stage biopsy, resulted in a birth of healthy twins born at 36 weeks' gestation. All the embryos were frozen after blastocyst biopsied. Totally, 54 frozen-thawed embryos that were transferred in 45 frozen-thawed embryo transfer cycles included 25 embryo from cleavage stage biopsy and 29 embryo from blastocyst stage biopsy, and 42 of them were HLA matched. Clinical pregnancy rate and implantation rate per cycle in frozen-thawed embryo transfer were 38%(17/45)and 37%(20/54)respectively. A total of 15 infants were born, 2 were from a fresh embryo transfer cycle, and 13 were from frozen-thawed embryo transfer cycles. RESULTS of prenatal diagnosis from delivered cases were matched to that of PGD. Four sick children have been cured by HSCT from these HLA matched born siblings. PGD with HLA matching is an established method for conceiving a child who may donate hematopoietic stem cells to save an ill sibling.

Obstetric and neonatal outcomes in blastocyst-stage biopsy with frozen embryo transfer and cleavage-stage biopsy with fresh embryo transfer after preimplantation genetic diagnosis/screening

To study whether embryo biopsy for preimplantation genetic diagnosis/preimplantation genetic screening (PGD/PGS) can influence pregnancy complications and neonatal outcomes. Retrospective analysis. University-affiliated center. This study included data from women and their neonates born after PGD/PGS (n = 317). Questionnaires were designed to obtain information relating to pregnancy complications and neonatal outcomes. Two major strategies for PGD/PGS were evaluated. Blastocyst-stage biopsy and frozen embryo transfer (BB-FET) was carried out in 166 patients, and cleavage-stage biopsy and fresh embryo transfer (CB-ET) was carried out in 129 patients. The incidence of gestational hypertension was significantly higher in BB-FET compared with in CB-ET (9.0% vs. 2.3%, adjusted odds ratio [OR] and 95% confidence interval [CI], 4.85 [1.34, 17.56]). In twins, the birthweight (median [range], 2.70kg [1.55-3.60kg] vs. 2.50kg [1.23-3.75kg]) was higher in BB-FET than in CB-ET and the gestational age was longer in BB-FET than in CB-ET (median [range], 36.71weeks [31.14-39.29weeks] vs. 35.57weeks [30.57-38.43weeks]). There was no difference in the incidence of singleton births between the two groups except in the incidence of preterm births (28-37weeks; 5.3% vs. 16.5% in CB-ET and BB-FET). No significant differences were detected in the incidence of perinatal deaths, birth defects, gender of neonates, and large for gestational age in both singletons and twins, although the numbers of some events were small. BB-FET is associated with a higher incidence of gestational hypertension but better neonatal outcomes compared with CB-ET, especially in twins.

The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis.

Preimplantation Genetic Diagnosis and Screening (PGD/PGS) for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying nonaffected and/or euploid embryos in a cohort produced during an IVF cycle. A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed. Cleavage stage blastomere biopsy still represents the most commonly used method in Europe nowadays, although this approach has been shown to have a negative impact on embryo viability and implantation potential. Polar body biopsy has been proposed as an alternative to embryo biopsy especially for aneuploidy testing. However, to date no sufficiently powered study has clarified the impact of this procedure on embryo reproductive competence. Blastocyst stage biopsy represents nowadays the safest approach not to impact embryo implantation potential. For this reason, as well as for the evidences of a higher consistency of the molecular analysis when performed on trophectoderm cells, blastocyst biopsy implementation is gradually increasing worldwide. The aim of this review is to present the evidences published to date on the impact of the biopsy at different stages of preimplantation development upon human embryos reproductive potential.

Reduction of multiple pregnancies in the advanced maternal age population after implementation of an elective single embryo transfer policy coupled with enhanced embryo selection: pre- and post-intervention study.

STUDY QUESTIONIs an elective single-embryo transfer (eSET) policy an efficient approach for women aged >35 years when embryo selection is enhanced via blastocyst culture and preimplantation genetic screening (PGS)?SUMMARY ANSWERElective SET coupled with enhanced embryo selection using PGS in women older than 35 years reduced the multiple pregnancy rates while maintaining the cumulative success rate of the IVF programme.WHAT IS KNOWN ALREADYMultiple pregnancies mean an increased risk of premature birth and perinatal death and occur mainly in older patients when multiple embryos are transferred to increase the chance of pregnancy. A SET policy is usually recommended in cases of good prognosis patients, but no general consensus has been reached for SET application in the advanced maternal age (AMA) population, defined as women older than 35 years. Our objective was to evaluate the results in terms of efficacy, efficiency and safety of an eSET policy coupled with increased application of blastocyst culture and PGS for this population of patients in our IVF programme.STUDY DESIGN, SIZE, DURATIONIn January 2013, a multidisciplinary intervention involving optimization of embryo selection procedure and introduction of an eSET policy in an AMA population of women was implemented. This is a retrospective 4-year (January 2010–December 2013) pre- and post-intervention analysis, including 1161 and 499 patients in the pre- and post-intervention period, respectively. The primary outcome measures were the cumulative delivery rate (DR) per oocyte retrieval cycle and multiple DR.PARTICIPANTS/MATERIALS, SETTING, METHODSSurplus oocytes and/or embryos were vitrified during the entire study period. In the post-intervention period, all couples with good quality embryos and less than two previous implantation failures were offered eSET. Embryo selection was enhanced by blastocyst culture and PGS (blastocyst stage biopsy and 24-chromosomal screening). Elective SET was also applied in cryopreservation cycles.MAIN RESULTS AND THE ROLE OF CHANCEPatient and cycle characteristics were similar in the pre- and post-intervention groups [mean (SD) female age: 39.6 ± 2.1 and 39.4 ± 2.2 years; range 36–44] as assessed by logistic regression. A total of 1609 versus 574 oocyte retrievals, 937 versus 350 embryo warming and 138 versus 27 oocyte warming cycles were performed in the pre- and post-intervention periods, respectively, resulting in 1854 and 508 embryo transfers, respectively. In the post-intervention period, 289 cycles were blastocyst stage with (n = 182) or without PGS (n = 107). A mean (SD) number of 2.9 ± 1.1 (range 1–4) and 1.4 ± 0.8 (range 1–3) embryos were transferred pre- and post-intervention, respectively (P < 0.01) and similar cumulative clinical pregnancy rates per transfer and per cycle were obtained: 26.8, 30.9% and 29.7, 26.3%, respectively. The total DR per oocyte retrieval cycle (21.0 and 20.4% pre- and post-intervention, respectively) defined as efficacy was not affected by the intervention [odds ratio (OR) = 0.8, 95% confidence interval (CI) = 0.7–1.1; P = 0.23]. However, a significantly increased live birth rate per transferred embryo (defined as efficiency) was observed in the post-intervention group 17.0 versus 10.6% (P < 0.01). Multiple DRs decreased from 21.0 in the preintervention to 6.8% in the post-intervention group (OR = 0.3. 95% CI = 0.1–0.7; P < 0.01).LIMITATIONS, REASONS FOR CAUTIONIn this study, the suitability of SET was assessed in individual women on the basis of both clinical and embryological prognostic factors and was not standardized. For the described eSET strategy coupled with an enhanced embryo selection policy, an optimized culture system, cryopreservation and aneuploidy screening programme is necessary.WIDER IMPLICATIONS OF THE FINDINGSOwing to the increased maternal morbidity and perinatal complications related to multiple pregnancies, it is recommended to extend the eSET policy to the AMA population. As shown in this study, enhanced embryo selection procedures might allow a reduction in the number of embryos transferred and the number of transfers to be performed without affecting the total efficacy of the treatment but increasing efficiency and safety.STUDY FUNDING/COMPETING INTEREST(S)None.TRIAL REGISTRATION NUMBERNone.

Birth of a healthy infant after preimplantation genetic diagnosis by sequential blastomere and trophectoderm biopsy for β-thalassemia and HLA genotyping

BackgroundPreimplantation genetic diagnosis (PGD) is a widely used technique for couples at genetic risk and involves the diagnosis and transfer of unaffected embryos generated through in vitro fertilization (IVF) techniques. Study designFor those couples who are at risk of transmitting a genetic disease to their offspring, preimplantation embryos can be selected according to their genetic status as well as human leukocyte antigen (HLA) compatibility with the affected child. Stem cells from the resulting baby's umbilical cord blood can be used for transplantation to the affected sibling without graft rejection. ResultsHere we report successful hematopoietic stem cell transplantation (HSCT) after the birth of a healthy infant, who was born after successful PGD testing with both cleavage stage and blastocyst stage biopsy for the purpose of diagnosis of β-thalassemia and HLA compatibility. ConclusionThe specific feature of this work is not only to have the first successful HSCT achieved in Bulgaria after using preimplantation HLA typing technique, it also demonstrates how to accomplish this success via cross-border collaboration of different units, which makes the application of these sophisticated methods possible in hospitals not having the necessary equipments and expertise.

Preimplantation genetic diagnosis and the biopsy technique: Important considerations

Preimplantation genetic diagnosis allows to test the genetic status of embryos prior to implantation. In order to obtain genetic material, on which carry out a genetic diagnosis, a procedure named embryo biopsy is required. In the last two decades, embryo biopsy at the cleavage stage has been the mostly performed procedure. However, recently, alternative methods allowing the retrieval of a larger number of cells (blastocyst stage biopsy), or representing a valid alternative to overcome ethical issues (polar body biopsy) have obtained increasing consensus. This article reviews different methods of embryo biopsy and points out their positive and negative aspects.

New Perspectives on Embryo Biopsy: Not How, But When and Why?

During the past 2 decades, biopsy for preimplantation genetic diagnosis at three stages, that is, before conception (the first polar body), after fertilization (the second polar body), and early cleavage (D3 blastomeres) or blastocyst stage (D5/D6 trophectoderm biopsy), have been optimized and performed clinically in hundreds of in vitro fertilization centers around the world. Although opening the zona pellucida by mechanical or chemical means is still effectively in use, noncontact laser has become the indispensable instrument. Overall, the invasive nature of biopsy at any given stage is recognized. It is believed that removal of the polar bodies from M-II oocytes and fertilized zygotes may have the least detrimental effects on subsequent embryonic development; hence increasing applications of polar body biopsy are anticipated. Although D3 biopsy is currently the most frequently used method, the effectiveness of D3 cleavage-stage biopsy is unsettling because of the mosaicism in early cleavage human embryos. Controversies exist in several areas; particularly, the efficacy of preimplantation genetic screening based on one cell removed from a D3 embryo remains to be confirmed. With new genetic testing technology, there may be no need to biopsy two cells because accuracy from one cell is high and the risk of misdiagnosis is very low when sufficient markers are used for chromosome copy number assessment or for mutation detection of single-gene disorders. And finally, it appears that limited harm is caused by biopsy at the blastocyst stage and mosaicism seems to be less common as compared with earlier stages. Therefore, use of the blastocyst-stage biopsy combined with cryopreservation protocol can be effectively used for several indications. Furthermore, faster genetic analytical methods that can be completed within several hours will make this strategy more practical and attractive as fresh embryo transfer is possible.

Efficiency of preimplantation genetic screening (PGS) using array-CGH compared to matched control IVF patient populations with and without day-3 PGS fish

Efficiency of preimplantation genetic screening (PGS) using array-CGH compared to matched control IVF patient populations with and without day-3 PGS fish