Intention to treat: how many initiated PGS cycles produce blastocysts for biopsy as maternal age increases?

Ovarian stimulation (OS) remains a crucial step of IVF process since cycle outcomes per cycle after OS are remarkably higher when compared to a natural or modified natural cycle. Within the concept of OS, two approaches are purposed: On one hand, “mild” stimulation claims for the use of low doses of gonadotrophins in order to yield a moderate number of eggs that should be enough to achieve a pregnancy. On the other, the so called “conventional” stimulation aims to maximize ovarian response, with the goal of obtaining the higher number of embryos that is possible and therefore increase the number of attempts per pick up to reach the highest chance of pregnancy for each patient in one cycle. Defenders of mild stimulation, claim that obtaining oocytes beyond a certain response (classically eight eggs) is useless, since these surplus oocytes won’t be able to provide good quality embryos. In other words, this statement is based in a hypothesis according to which a soft stimulation of the ovaries would lead them to mature only the good quality eggs, while those of lower quality would not respond to these low doses and would remain immature in the ovaries. A few numbers of old studies performed with outdated technologies support this this concept. However, a large body of evidence shows that the contrary is true. Old studies that related ovarian response to cycle outcome considering only the chances of achieving a live birth after just a fresh embryo transfer, showed that live birth rate increased as the ovarian response did up to around 15 oocytes, while it shows a “plateau” beyond this response. But moreover, more recent studies in which the cumulative live birth rate achieved after one live birth is achieved or until all viable embryos that were cryopreserved were transferred, shows that it improves linearly as the ovarian response does, starting to plateau only for ovarian responses larger than 35-40 eggs. A very recent study that analyses more than 400,000 cycles from the Society for Assisted Reproductive Technology Clinic Outcome Reporting System (SART) confirms these figures. This large sample allows for stratified analysis according to age, BMI, AMH or infertility diagnoses that further confirm the findings. Altogether, these studies show that the concept of some kind of “oocyte quality selection” happens when a mild stimulation is used does not really happen. Large ovarian responses increase the number of good and bad quality oocytes equally. The idea of ovarian stimulation hampering oocyte quality has been around for more than 30 years. Theorical concepts based on observations in the animal model suggested that this might be the case. However, studies in humans have been unable to prove this hypothesis. In this context, the best model to prove this concept is the intra-patient comparison between the non-stimulated and the conventionally stimulated cycle. Our group has performed two studies following this approach so far. In the first one, we included 46 oocyte donors. PGT-A was performed to the embryos obtained in both cycles, which at time was performed on day 3 embryos with FISH technology. The study showed that there were no significant differences in the aneuploidy rate between the 2 cohorts of embryos (34.8% vs 32.8%), suggesting no harm of ovarian stimulation. These findings have been recently confirmed in 40 infertile patients aged 30-38, in which PGT-A was done with a comprehensive chromosomal analysis using NGS after blastocyst biopsy. In conclusion, research and clinical evidence show that conventional OS does not harm oocyte quality and increases the cumulative birth rate per oocyte pick-up, making it as the first choice for treating patients with IVF. Trial registration number XXXX

Deep impact: sequencing embryo biopsy specimens at increasing depth

STUDY QUESTION Can quantification of mitochondrial DNA (mtDNA) in trophectoderm (TE) biopsy samples provide information concerning the viability of a blastocyst, potentially enhancing embryo selection and improving IVF treatment outcomes? SUMMARY ANSWER This study demonstrated that euploid blastocysts of good morphology, but with high mtDNA levels had a greatly reduced implantation potential. WHAT IS KNOWN ALREADY Better methods of embryo selection leading to IVF outcome improvement are necessary, as the transfer of chromosomally normal embryos of high morphological grade cannot guarantee the establishment of an ongoing pregnancy. The quantity of mtDNA in embryonic cells has been proposed as a new biomarker of viability-higher levels of mtDNA associated with reduced implantation potential. STUDY DESIGN, SIZE, DURATION mtDNA was quantified in 199 blastocysts, previously biopsied and shown to be chromosomally normal using preimplantation genetic testing for aneuploidy (PGT-A). These were generated by 174 couples (average female age 37.06 years). All patients underwent IVF in a single clinic. The study took place in a blinded, non-selection manner-i.e. mtDNA quantity was not known at the time of single embryo transfer. The fate of the embryos transferred was subsequently compared to the mtDNA levels measured. PARTICIPANTS/MATERIALS, SETTING, METHODS Embryos were biopsied at the blastocyst stage. The TE samples obtained were subjected to whole genome amplification followed by comprehensive chromosome analysis via next generation sequencing. The same biopsy specimens were also tested using quantitative PCR, allowing highly accurate mtDNA quantification. After blastocyst transfer, the code used for blinding was broken and analysis undertaken to reveal whether the amount of mtDNA had any association with embryo implantation. MAIN RESULTS AND THE ROLE OF CHANCE mtDNA analysis of the 199 blastocysts revealed that 9 (5%) contained unusually high levels of mtDNA. All embryo transfers involved a single chromosomally normal blastocyst of good morphology. Of these, 121 (60%) led to ongoing pregnancies, 11(6%) led to biochemical pregnancies, and 10 (5%) spontaneously miscarried. All (100%) of these blastocysts had mtDNA levels considered to be normal/low. The remaining 57 (29%) blastocysts failed to implant. Among these non-viable embryos there were 9 (16%) with unusually high levels of mtDNA. This meant that the ongoing pregnancy rate for morphologically good, euploid blastocysts, with normal/low levels of mtDNA was 64% (121/190). In contrast, the ongoing pregnancy rate for the same type of embryos, but with elevated mtDNA levels, was 0/9 (0%). This difference was highly statistically significant (P < 0.0001). LIMITATIONS REASONS FOR CAUTION To determine the true extent of any clinical benefits a randomized clinical trial will be necessary. Research is needed to improve understanding of the biology of mtDNA expansion. WIDER IMPLICATIONS OF THE FINDINGS This is the first investigation to evaluate the clinical impact of increased mtDNA in a prospective blinded manner. Results confirm that embryos with elevated mtDNA rarely implant, supporting its use as a viability biomarker. A total of 64% of euploid blastocysts with normal/low mtDNA implanted versus 60% for the cohort as a whole. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by institutional funding (Reprogenetics UK and Reprogenetics). DW is supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. None of the authors have any competing interests.

Chapter 29 - Preimplantation genetic testing

Preimplantation genetic diagnosis (PGD) is gradually widely used in prevention of gene diseases and chromosomal abnormalities. Much improvement has been achieved in biopsy technique and molecular diagnosis. Blastocyst biopsy can increase diagnostic accuracy and reduce allele dropout. It is cost-effective and currently plays an important role. Whole genome amplification permits subsequent individual detection of multiple gene loci and screening all 23 pairs of chromosomes. For PGD of chromosomal translocation, fluorescence in-situ hybridization (FISH) is traditionally used, but with technical difficulty. Array comparative genomic hybridization (CGH) can detect translocation and 23 pairs of chromosomes that may replace FISH. Single nucleotide polymorphisms array with haplotyping can further distinguish between normal chromosomes and balanced translocation. PGD may shorten time to conceive and reduce miscarriage for patients with chromosomal translocation. PGD has a potential value for mitochondrial diseases. Preimplantation genetic haplotyping has been applied for unknown mutation sites of single gene disease. Preimplantation genetic screening (PGS) using limited FISH probes in the cleavage-stage embryo did not increase live birth rates for patients with advanced maternal age, unexplained recurrent abortions, and repeated implantation failure. Polar body and blastocyst biopsy may circumvent the problem of mosaicism. PGS using blastocyst biopsy and array CGH is encouraging and merit further studies. Cryopreservation of biopsied blastocysts instead of fresh transfer permits sufficient time for transportation and genetic analysis. Cryopreservation of embryos may avoid ovarian hyperstimulation syndrome and possible suboptimal endometrium.

Validation of microarray comparative genomic hybridization for comprehensive chromosome analysis of embryos

15.IMPACT OF MATERNAL AGE IN THE INCIDENCE OF UNIFORM AND MOSAIC ANEUPLOIDIES

To study reproductive outcomes of transferring embryos with mosaic chromosomal aneuploidies unrelated to target loci in preimplantation genetic testing for structural rearrangement (PGT-SR). A retrospective analysis of 1712 PGT-SR cycles (involving 1373 couples) from a single center was conducted. Comprehensive chromosomal analysis was performed on trophectoderm biopsy samples using whole-genome amplification combined with next-generation sequencing. Clinical outcomes after single embryo transfer, including clinical pregnancy rate, miscarriage rate, live birth rate, and ectopic pregnancy rate, were compared between mosaic embryo transfers and completely euploid embryo transfers. In PGT-SR cycles, euploid embryo transfers (1338 cycles) demonstrated a clinical pregnancy rate of 65.17%, live birth rate of 58.67%, miscarriage rate of 6.05%, and ectopic pregnancy rate of 0.45%, whereas mosaic embryo transfers (165 cycles) exhibited comparable outcomes with 58.18%, 54.55%, 3.64%, and 0%, respectively. The subgroup analysis by mosaic degree which revealed low-level mosaicism (20%-29% and 30%-49%) had outcomes similar to euploid embryos. High-level mosaicism (50%-69%) demonstrated significantly reduced clinical pregnancy (OR = 0.20, 95% CI 0.06-0.54) and live birth rates (OR = 0.20, 95% CI 0.06-0.56), though miscarriage remained comparable. No significant outcome variations were observed across chromosomal types. In PGT-SR cycles, embryos with low-level mosaicism (20%-49%) unrelated to target loci demonstrate equivalent clinical pregnancy and live birth outcomes to completely euploid embryos. Although high-level mosaicism (50%-69%) is likely associated with reduced live birth rates, these embryos still retain potential for healthy live births.

Study question Does the selection of blastocysts for single embryo transfer, through the diagnosis of aneuploidy, improves the live birth rate in patients undergoing in vitro fertilization? Summary answer There seems to be no statistical difference in live birth rates between embryos with preimplantational genetic diagnosis (PGD) and those without. What is known already: Initial reports indicate that reproductive results improve after the selection of embryos to be transfer after performing a biopsy of the blastomeres, or trophectoderm cells, with the subsequent comprehensive analysis of the chromosomes. However, these results are now questioned. Reports in the literature are contrasting, so the real utility of selecting all embryos through comprehensive chromosome analysis calls for a more careful analysis that compares the risks, costs, and benefits of these techniques and their actual utility in reproductive results of patients treated with in vitro fertilization. Specifically results related to live birth rate. Study design, size, duration A systematic review of prospective studies evaluating live birth rate after embryo transfer of embryos selected by blastocyst biopsy for aneuploidy analysis compared with reproductive outcomes in embryo transfers of embryos selected morphologically, without biopsy nor screening for aneuploidies. Participants/materials, setting, methods A literature search was performed in PubMed, EmBase, and the Cochrane library (from January 2000 to december 2019). A cumulative meta-analysis and evaluation of heterogeneity was performed for the clinical pregnancy rate. The quality of the included studies was assessed using Cochrane’s Risk of Bias tool and ROBINS I for observational studies Main results and the role of chance Seven studies were included, three were randomized controlled trials and four were non-randomized studies of intervention (NRSI). The included studies were published between 2013 and 2019. For the preimplantational genetic diagnosis, three studies used array comparative genomic hybridization, three studies used next generation sequencing and only one study used qPCR. A total of 1638 patients were included, only two studies excluded patients with advanced maternal age (&amp;gt;35 years), two studies studied patients with recurrent implantation failure and three studies patients with recurrent pregnancy loss. Regarding the assisted reproduction techniques (ART), only studies where embryos where biopsied after day five for the genetic diagnosis where considered, most used ICSI and performed frozen-thawed transfer of up to two embryos, only one study allowed patients to be transferred with more than two embryos per cycle. Reproductive outcomes (live birth rate, miscarriage rate, clinical pregnancy) were extracted considering the events per embryo transfer and calculating the pooled odds ratios (OR) with 95% confidence intervals (95%CI) as our main outcome, sensitivity analyses will be performed using the events per cycles to assess the robustness of the effect estimate. Preliminary meta-analyses resulted in a pooled OR of 1.45 (95%CI 0.24–8.78) for NRSI and 1.34 (95%CI 0.85–2.11) for RCT. Limitations, reasons for caution The main limitation was the quantity of studies with acceptable methodology. This generated heterogeneity, hindering the evaluation of the true impact of PGD in ART outcomes. The use of events per embryo transfer as a main outcome could bias the results favoring PGD as less embryos are usually transferred. Wider implications of the findings: Our results show that there are too few studies with adequate methodology to generate a conclusion about the true benefit of PGD. However, a slight tendency favoring the reproductive outcomes of PGD was found. Trial registration number PROSPERO CRD42020198866

PurposeThe aim of this study is to investigate the minimum number of blastocysts for biopsy to increase the likelihood of obtaining at least one normal/balanced embryo in preimplantation genetic diagnosis (PGD) for translocation carriers.MethodsThis blinded retrospective study included 55 PGD cycles for Robertsonian translocation (RT) and 181 cycles for reciprocal translocation (rcp) to indicate when only one of the couples carried a translocation. Single-nucleotide polymorphism microarray after trophectoderm biopsy was performed.ResultsReliable results were obtained for 355/379 (93.7 %) biopsied blastocysts in RT group and 986/1053 (93.6 %) in rcp group. Mean numbers of biopsied embryos per patient, normal/balanced embryos per patient, and mean normal/balanced embryo rate per patient were 7.4, 3.1, and 40.7 % in RT group and 8.0, 2.1, and 27.3 %, respectively, in rcp group. In a regression model, three factors significantly affected the number of genetically transferrable embryos: number of biopsied embryos (P = 0.001), basal FSH level (P = 0.040), and maternal age (P = 0.027). ROC analysis with a cutoff of 1.5 was calculated for the number of biopsied embryos required to obtain at least one normal/balanced embryo for RT carriers. For rcp carriers, the cutoff was 3.5. The clinical pregnancy rate per embryo transfer was 44.2 and 42.6 % in RT and rcp groups (P = 0.836).ConclusionsThe minimum numbers of blastocysts to obtain at least one normal/balanced embryo for RT and rcp were 2 and 4 under the conditions of female age < 37 years with a basal FSH level < 11.4 IU/L.

Investigate the chromosome status and transfer outcomes of embryos selected using routine "best morphology" IVF practices. A prospective multi-center, non-selection cohort study involving patients undertaking IVF treatment. Study entry conditions were blastocyst biopsy, >1 embryo with chromosome analysis and frozen transfer of the best morphology embryo. Primary analyses were βhCG positive, implantation, ongoing pregnancy and birth rates and pregnancy-stage progression failures. After transfer, embryo chromosome status was assigned and outcomes divided into two primary groups - euploids (n=135) and aneuploids (n=53). Compared to euploid embryo transfers, aneuploid embryos had significantly lower primary outcomes (+βhCG: 67% vs. 30%, p<0.0001; IR: 56% vs. 19%, p<0.0001; ongoing week 12: 51% vs. 9%, p<0.0001; and livebirths: 50% vs. 8%, p<0.0001, respectively). Transfers were further subdivided into smaller groups according to their main chromosomal feature. Stage analysis showed higher failure rates for aneuploids to initiate a pregnancy (p<0.0001), higher subclinical miscarriage rate (p=0.0402) and higher clinical miscarriage rate (p=0.0038). Routine morphology-based embryo selection resulted in a high euploid selection rate but a significant number of aneuploid embryos were still inadvertently selected for transfer (28%) with the subsequent high failure rates for pregnancy initiation and progression having implications for appropriate patient management.

Defining assisted reproductive technology success

Two different microarray technologies for preimplantation genetic diagnosis (PGD) and screening (PGS), due to reciprocal translocation imbalances, demonstrate equivalent clinical pregnancy rates

Preimplantation genetic diagnosis and screening are widely accepted for chromosomal abnormality identification to avoid transferring embryos with genetic defects. Massively parallel sequencing (MPS) is a rapidly developing approach for genome analysis with increasing application in clinical practice. The purpose of this study was to use MPS for identification of aneuploidies and unbalanced chromosomal rearrangements after blastocyst biopsy. Trophectoderm (TE) samples of 38 blastocysts from 16 in vitro fertilization cycles were subjected to analysis. Low-coverage whole genome sequencing was performed using the Illumina HiSeq2000 platform with a novel algorithm purposely created for chromosomal analysis. The efficiency of this MPS approach was estimated by comparing results obtained by an Affymetrix single-nucleotide polymorphism (SNP) array. Whole genome amplification (WGA) products of TE cells were detected by MPS, with an average of 0.07× depth and 5.5% coverage of the human genome. Twenty-six embryos (68.4%) were detected as euploid, while six embryos (15.8%) contained uniform aneuploidies. Four of these (10.5%) were with solely unbalanced chromosomal rearrangements, whereas the remaining two embryos (5.3%) showed both aneuploidies and unbalanced rearrangements. Almost all these results were confirmed by the SNP array, with the exception of one sample, where different sizes of unbalanced rearrangements were detected, possibly due to chromosomal GC bias in array analysis. Our study demonstrated MPS could be applied to accurately detect embryonic chromosomal abnormality with a flexible and cost-effective strategy and higher potential accuracy.

298 Background: Although FLOT (5-fluorouracil [5-FU]/leucovorin [ l-LV] + oxaliplatin [L-OHP] + docetaxel [DTX]) is globally recognized as the standard treatment for gastric cancer (GC), the safety of FLOT in Japanese patients (pts) is unknown. Moreover, because advanced GC (AGC) pts with severe peritoneal metastasis (SPM) have often been excluded from pivotal clinical trials due to their poor condition, it is necessary to develop standardized treatment for them. Methods: This study is an open-label, multicenter, single-arm, phase I study to determine the recommended phase II dose (RP2D) of FLOT for Japanese AGC pts without (part 1) or with (part 2) SPM using a 3 + 3 design. Main eligibility criteria included histologically confirmed AGC, HER2 negative, and age 20–75 years. In part 2a, pts with ECOG Performance status (PS) 2, who simultaneously had massive ascites and inadequate oral intake, were excluded. Massive ascites was defined as continuous ascites from the pelvic cavity to the upper abdomen, based on computed tomography findings. Inadequate oral intake was defined as the requirement for daily intravenous infusions to supply water or nutrition. The dose limiting toxicity (DLT) was assessed before the start of cycle 2. The fixed dose of L-OHP, l-LV, and 5-FU was 85, 200, and 2600 mg/m2, respectively, and DTX was administered at a dose of 50, 40, and 30 mg/m2 in the level 0, −1, and −2, respectively. Results: In part 1a, one patient died of hemorrhage from the primary tumor on Cycle 1 Day 2 and was replaced. The characteristics were as follows: median age (range), 70 (51–71) years; male/female, 2/1; ECOG PS (0/1), 2/1; histological type (diffuse/intestinal), 3/0; prior gastrectomy (yes/no), 0/3; and measurable lesions (yes/no), 1/2. During DLT evaluation, grade 3 (G3) or higher adverse events (AEs) were neutropenia (n = 1), lymphocytopenia (n = 1), anemia (n = 1), and anorexia (n = 1). During follow-up, the most common G3 or higher AEs included neutropenia (n = 3), lymphocytopenia (n = 1), anemia (n = 1), febrile neutropenia (FN) (n = 1 at cycle 5), diarrhea (n = 1), anorexia (n = 1), fatigue (n = 1), and colonic perforation (n = 1) due to diverticulitis (not treatment-related AE). In part 2a, the characteristics were as follows: median age (range) 64 (49–69) years; male/female, 3/0; ECOG PS (0/1), 0/3; histological type (diffuse/intestinal), 3/0; prior gastrectomy (yes/no), 0/3; and measurable lesions (yes/no), 0/3. Two pts had massive ascites and 1 pt had both massive ascites and inadequate oral intake. During follow-up, only G3 neutropenia (n = 3) was observed. Two pts needed dose modification in cycle 2. Both in part 1a and 2a, no treatment-related deaths and no DLTs were observed and the RP2D was determined in level 0. Conclusions: We determined that DTX 50 mg/m2, L-OHP 85 mg/m2, 5-FU 2600 mg/m2, and l-LV 200 mg/m2 were the RP2D of FLOT for Japanese AGC with/without SPM. Clinical trial information: 041200044.