The effect of vitrification on blastocyst mitochondrial DNA dynamics and gene expression profiles.

PURPOSE: Does vitrification/warming affect the mitochondrial DNA (mtDNA) content and the gene expression profile of blastocysts? METHODS: Prospective cohort study in which 89 blastocysts were obtained from 50 patients between July 2017 and August 2018. mtDNA was measured in a total of 71 aneuploid blastocysts by means of real-time polymerase chain reaction (RT-PCR). Transcriptomic analysis was performed by RNA sequencing (RNA-seq) in an additional 8 aneuploid blastocysts cultured for 0 h after warming, and 10 aneuploid blastocysts cultured for 4-5 h after warming. RESULTS: A significant decrease in mtDNA content just during the first hour after the warming process in blastocysts was found (P < 0.05). However, mtDNA content experimented a significantly increased along the later culture hours achieving the original mtDNA levels before vitrification after 4-5 h of culture (P < 0.05). Gene expression analysis and functional enrichment analysis revealed that such recovery was accompanied by upregulation of pathways associated with embryo developmental capacity and uterine embryo development. Interestingly, the significant increase in mtDNA content observed in blastocysts just after warming also coincided with the differential expression of several cellular stress response-related pathways, such as apoptosis, DNA damage, humoral immune responses, and cancer. CONCLUSION: To our knowledge, this is the first study demonstrating in humans, a modulation in blastocysts mtDNA content in response to vitrification and warming. These results will be useful in understanding which pathways and mechanisms may be activated in human blastocysts following vitrification and warming before a transfer.

Elevated serum progesterone does not impact euploidy rates in PGT-A patients.

PURPOSE: Some women undergoing stimulated cycles have elevated serum progesterone (P) on the day of ovulation trigger, but its effect on embryo quality is unclear. We analyze embryo quality among patients with high and low serum P undergoing preimplantation genetic testing for aneuploidy (PGT-A). METHODS: This retrospective study included 1597 patients divided into two groups by serum P values: < 1.5 ng/mL or ≥ 1.5 ng/mL. A gonadotrophin-releasing hormone (GnRH) antagonist protocol was established for each patient. Serum P levels were measured on the day of triggering. Propensity score matching and Poisson regression were done. Age, body mass index, and ovarian sensitivity index were also compared. RESULTS: Elevated serum P was not significantly associated with euploid embryo rate or other embryo-quality variables evaluated in our study. Age was the only variable associated with euploidy rate (per MII oocyte, P < 0.001; per biopsied embryo, P = 0.008), embryo biopsy rate (P < 0.001), absolute number of euploid embryos (P = 0.008), and top-quality embryo rate (P = 0.008). Categorical variables decreased in value for every year of increased age in patients with high serum P. CONCLUSIONS: Elevated serum P did not affect the number of euploid and good-quality embryos for transfer in GnRH antagonist intracytoplasmic sperm injection (ICSI) cycles. Contrary to the clear influence of premature P elevation on endometrial receptivity based on literature, our results may help to tip the balance towards the absence of a negative effect of P elevation on embryo competence. More studies are needed to fully understand the effect of P elevation on reproductive outcomes.

The morphokinetic signature of mosaic embryos: evidence in support of their own genetic identity.

OBJECTIVE: To provide full morphokinetic characterization of embryos ranked with different degrees of chromosomal mosaicism. DESIGN: Retrospective cohort study. SETTING: University-affiliated private in vitro fertilization clinic. PATIENT(S): We analyzed 1,511 embryos from 424 intracytoplasmic sperm injection cycles by culturing embryos in a time-lapse imaging system and performing next-generation sequencing. We assessed 106 mosaic embryos. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Comparison of chromosomal, morphological, and morphokinetic characteristics of blastocysts classified as euploid, aneuploid, low-degree mosaic (30% to <50% aneuploid cells in trophectoderm biopsy), and high-degree mosaic (50% to <70% aneuploid cells in trophectoderm biopsy). Statistical analysis was performed using χ2, Kruskal-Wallis, or analysis of variance tests according to data type and distribution. A two-way random effects model was used to calculate interoperator correlation of annotations, and a logistic mixed effects model was performed to evaluate the effect of confounders on morphokinetic timing. RESULT(S): The mosaicism rate was ∼7% regardless of parental age. Mosaicism and uniform aneuploidies were not evenly distributed across chromosomes. The percentage of high-quality blastocysts significantly decreased from euploid (66.9%) to mosaic (52.8%) and aneuploid (47.7%). Aneuploid blastocysts significantly delayed development compared with euploid blastocysts in start of compaction (median, 84.72 hours postmicroinjection [hpm], interquartile range [IQR], 13.2; vs. median, 82.10 hpm, IQR, 11.5), start of blastulation (median, 101 hpm; IQR, 11.7; vs. median, 98.29 hpm, IQR, 10.5), and timing of blastocyst (median, 108.04 hpm, IQR, 11.50; vs. median, 104.71 hpm, IQR, 11.35). However, embryo morphokinetics were not correlated to the degree of mosaicism or to a mosaicism configuration that was apt for embryo transfer. CONCLUSION(S): Morphokinetic timing of mosaic embryos overlaps with that of euploid and aneuploid embryos, which may reflect their unique genetic and developmental identity. Although this suggests mosaic embryos are not simply a misdiagnosis by-product, further studies are needed to reveal the true identity of this particular type of embryo.

Mitochondrial DNA content decreases during in vitro human embryo development: insights into mitochondrial DNA variation in preimplantation embryos donated for research.

OBJECTIVE: To assess the mitochondrial DNA (mtDNA) load and variation in human oocytes and during preimplantation embryo development using specimens donated for research. DESIGN: Prospective cohort study. SETTING: Not applicable. PATIENTS: A total of 50 in vitro fertilization patients and 11 oocyte donors whose specimens were obtained between July 2017 and July 2018. INTERVENTIONS: None. MAIN OUTCOME MEASURES: All specimens were separately collected. Quantitative polymerase chain reaction was performed with SurePlex DNA Amplification System (Illumina). Primers for the adenosine triphosphate 8 mitochondrial gene and the ß-actin were used. Data were statistically analyzed by analysis of variance with the Scheffé multiple pairwise comparison for categorical variables and by linear regression for numerical variables. RESULTS: Human metaphase II (MII) oocytes had significantly more total mtDNA copy number than day 3 embryos, and day 3 embryos had more total and per-cell mtDNA copy number than aneuploid blastocysts. There was a significant decrease in mtDNA content associated with failed-fertilized oocytes compared to noninseminated metaphase II oocytes. CONCLUSIONS: During preimplantation development, before implantation, human embryos undergo a significant decrease in total mtDNA content and no increase in mtDNA content at the blastocyst stage. Oocytes need to carry a correct threshold of mitochondrial load in the oocyte in order to successfully fertilize. An active degradation of mtDNA before implantation occurs after fertilization takes place. These findings could be used to improve knowledge about the best embryo culture conditions and would serve as a basis for further studies addressing again the use of mtDNA content as an embryo viability marker.

Variables associated with mitochondrial copy number in human blastocysts: what can we learn from trophectoderm biopsies?

OBJECTIVE: To study the potential variables that affect the mitochondrial DNA (mtDNA) content of trophectoderm (TE) cells in blastocysts that have undergone TE biopsy. DESIGN: Observational retrospective single-center analysis. SETTING: University-affiliated private in vitro fertilization center. PATIENT(S): A total of 465 consecutive preimplantation genetic screening (PGS) cycles of 402 women undergoing preimplantation genetic testing. INTERVENTION(S): Trophectoderm biopsy performed on blastocysts of women undergoing preimplantation genetic testing-aneuploidy (PGT-A). MAIN OUTCOME MEASURE(S): The mtDNA content in trophectoderm cells. RESULT(S): We checked the possible influence of patient characteristics, ovarian stimulation variables, embryo morphology, and embryo culture conditions on mtDNA values. Of all the analyzed variables, some such as body mass index (BMI), serum progesterone (P4), aneuploidy, and trophectoderm quality had an effect on mtDNA content in blastocysts. Body mass index had a small but positive effect on the mtDNA copy number; as the BMI values increased, the probability of women producing blastocysts with an mtDNA content above the median increased by 6%. For P4 serum concentration, an increase in P4 lowered the probability of blastocysts having values above the median by 39%. Embryo-associated variables such as TE quality and aneuploidy status appeared to affect the mtDNA copy number. For the aneuploid blastocysts, the probability of being above the median increased by 42%. Finally, blastocysts with poor quality TE had more chances of carrying higher mtDNA values. CONCLUSION(S): Summarizing, larger quantities of mtDNA in blastocysts are associated with the condition of aneuploidy and low quality TE, as well as being from women with high BMI values. Understanding the biological meaning of mtDNA content in human blastocysts and what factors may interfere with their values is fundamental. Other key gaps, such as whether a correlation exists between mtDNA content and mitochondrial mass and biogenesis in human TE cells, and whether this correlation can be extended to the inner cell mass, need to be further addressed. These questions are currently being investigated.

A Higher Ovarian Response after Stimulation for IVF Is Related to a Higher Number of Euploid Embryos.

This study has analysed the relationship between ovarian response and the number of euploid embryos. This is a post hoc analysis of a subset of data generated during a prospective cohort study previously published. Forty-six oocyte donors were subjected to ovarian stimulation with 150 IU of rFSH and 75 IU of hp-hMG in a GnRH agonist long protocol. Preimplantation genetic screening was performed in all viable embryos. We observed a positive relationship between ovarian response and the number of euploid embryos. When ovarian response was above the median (≥17 oocytes), the mean number of euploid embryos per donor was 5.0 ± 2.4, while when <17 oocytes were obtained the mean number of euploid embryos was 2.7 ± 1.4 (p = 0.000). Aneuploidy rate did not increase with ovarian response or gonadotropin doses. Also, the number of euploid embryos was inversely related to the amount of gonadotropins needed per oocyte obtained (ovarian sensitivity index). These results suggest that the number of euploid embryos available for embryo transfer increases as the number of oocytes obtained does. Considering the total number of euploid embryos seems more relevant than the aneuploidy rate.

Distribution patterns of segmental aneuploidies in human blastocysts identified by next-generation sequencing.

OBJECTIVE: To evaluate the ability of next-generation sequencing (NGS) to detect pure and mosaic segmental aneuploidies in trophectoderm biopsies and to identify distribution patterns in whole blastocysts. DESIGN: Validation study. SETTING: Reference laboratory. PATIENT(S): Seventy couples with known karyotypes who had undergone preimplantation genetic screening with diagnoses at the blastocyst stage using array comparative genomic hybridization (aCGH). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Concordance rates for segmental and whole-chromosome aneuploidies determined between aCGH and NGS, and estimates of mosaicism levels of segmental aneuploidies in fixed blastocysts. RESULT(S): We used NGS with amplified DNA from trophectoderm biopsies in which segmental aneuploidies had been previously detected by array comparative genomic hybridization (aCGH). Single-cell fluorescent in situ hybridization (FISH) was then used as an independent form of analysis. The concordance rate between NGS and aCGH was 124 (98.4%) of 126 for the detection of segmental aneuploidies, and 48 (96.0%) of 50 for whole-chromosome aneuploidies. The overall concordance rate was 99.8% (2,276 of 2,280 chromosomes assessed). After FISH analyses with 41.4 ± 24.3 cells per blastocyst, 26 (92.9%) of 28 segmentals detected by aCGH and NGS were confirmed. The FISH analysis did not detect the segmentals in two blastocysts, in which all cells analyzed were euploid. CONCLUSION(S): This is the first report analyzing distribution patterns of segmental aneuploidies in trophectoderm biopsy by NGS. We have demonstrated that NGS allows the detection of pure and mosaic segmental aneuploidies with the same efficiency as aCGH. The FISH analysis confirmed the existence of these events in the trophectoderm and the inner cell mass.

New tools for embryo selection: comprehensive chromosome screening by array comparative genomic hybridization.

The objective of this study was to evaluate the usefulness of comprehensive chromosome screening (CCS) using array comparative genomic hybridization (aCGH). The study included 1420 CCS cycles for recurrent miscarriage (n = 203); repetitive implantation failure (n = 188); severe male factor (n = 116); previous trisomic pregnancy (n = 33); and advanced maternal age (n = 880). CCS was performed in cycles with fresh oocytes and embryos (n = 774); mixed cycles with fresh and vitrified oocytes (n = 320); mixed cycles with fresh and vitrified day-2 embryos (n = 235); and mixed cycles with fresh and vitrified day-3 embryos (n = 91). Day-3 embryo biopsy was performed and analyzed by aCGH followed by day-5 embryo transfer. Consistent implantation (range: 40.5-54.2%) and pregnancy rates per transfer (range: 46.0-62.9%) were obtained for all the indications and independently of the origin of the oocytes or embryos. However, a lower delivery rate per cycle was achieved in women aged over 40 years (18.1%) due to the higher percentage of aneuploid embryos (85.3%) and lower number of cycles with at least one euploid embryo available per transfer (40.3%). We concluded that aneuploidy is one of the major factors which affect embryo implantation.

Preimplantation genetic screening using fluorescence in situ hybridization in patients with repetitive implantation failure and advanced maternal age: two randomized trials.

OBJECTIVE: To evaluate the usefulness of preimplantation genetic screening (PGS) using fluorescence in situ hybridization (FISH) for two different indications: repetitive implantation failure (RIF) and advanced maternal age (AMA). DESIGN: Two prospective, randomized controlled trials with patients allocated in two arms: blastocyst transfer on day 5 (group A) or PGS with transfer on day 5 (group B). SETTING: University-affiliated private clinics. PATIENT(S): The RIF study included women <40 years with three or more failed IVF cycles without other known causal factors (91 patients). The AMA study included intracytoplasmic sperm injection patients aged between 41 and 44 (183 patients). INTERVENTION(S): In the PGS group, single-cell day 3 biopsy was performed with aneuploidy screening for chromosomes 13, 15, 16, 17, 18, 21, 22, X, and Y. In both the blastocyst transfer group and the PGS group, ET was performed on day 5. MAIN OUTCOME MEASURE(S): Live-birth rate per patient and per started cycle. RESULT(S): A significant increase in live-birth rates per patient was found in the PGS group compared with the blastocyst group for the AMA study (30/93 patients [32.3%] vs. 14/90 patients [15.5%]; odds ratio, 2.585; confidence interval, [1.262-5.295]). In the RIF study no significant differences were observed (23/48 patients [47.9%] vs. 12/43 patients [27.9%]). CONCLUSION(S): PGS with FISH was shown to be beneficial for the AMA group.

False positive rate of an arrayCGH platform for single-cell preimplantation genetic screening and subsequent clinical application on day-3.

In this work, false positive rate of an arrayCGH platform for its use in day-3 single-blastomere analysis was calculated. For this purpose, 38 embryos diagnosed as abnormal on day-3 by FISH were re-biopsied on day-4. Single-cell day-4 arrayCGH diagnosis was then performed. A successful amplification was obtained in 97.4 % (37/38) of the day-4 cells analysed by arrayCGH. Day-3 FISH and day-4 arrayCGH diagnosis were concordant in 35/37 cases. The two discordant embryos were spread and all the cells from each embryo were re-analysed by FISH on day 5. The same error rate (2.7 %) for day-3 FISH and day-4 arrayCGH was obtained when comparing day-5 FISH re-analysis. After this pre-clinical phase, the platform was used for day-3 arrayCGH clinical application in 320 patients (1,760 embryos). Day-3 amplification rate was 98.6 %. An optimal reproductive outcome was obtained when applying arrayCGH to a clinical program: clinical pregnancy rate per cycle of 38.4 % and 60.3 % per transference were obtained, with an implantation rate of 53.5 %. Overall miscarriage rate was 10.6 %. Additionally, day-5 FISH re-analysis was performed in 42 of the embryos from the clinical phase, obtaining a concordance rate of 97.6 % with day-3 arrayCGH.

Prospective cohort study in high responder oocyte donors using two hormonal stimulation protocols: impact on embryo aneuploidy and development.

BACKGROUND: Ovarian stimulation regimens for in vitro fertilization seem to have a deleterious effect on oocyte quality and embryo aneuploidy in a dose-dependent manner. This study aims to test the influence of gonadotrophin doses on embryo aneuploidy rates. METHODS: A total of 32 young oocyte donors with a high response to ovarian stimulation, were included in the study. Two subsequent stimulation treatments were performed in each donor: first, a standard dose cycle using a 225 IU starting dose of recombinant FSH (r-FSH) and secondly, a reduced dose cycle with a starting dose of 150 IU r-FSH. In both cycles, GnRH agonist co-treatment was used for down-regulation. Ovarian response, embryo development and aneuploidy for chromosomes 13, 15, 16, 17, 18, 21, 22, X and Y were the main outcomes of the study. RESULTS: A total of 22 donors completed both treatments with different gonadotrophin doses. In the remaining 10 donors, the reduced dose cycle was cancelled due to low ovarian response. In those donors who completed both regimens, significant increases in rates of fertilization and chromosomally normal blastocysts were observed in the reduced dose cycle. No differences were observed in pregnancy and implantation rates in recipients who received oocytes from standard and reduced doses cycles. CONCLUSIONS: Despite the limited numbers in our study, we can conclude that in high responder donors, a decrease in the gonadotrophin dose could improve fertilization rates and embryo quality. However, due to the reduced oocyte numbers with lower doses, a similar reproductive outcome in terms of live births would be expected.

Embryologic outcome and secretome profile of implanted blastocysts obtained after coculture in human endometrial epithelial cells versus the sequential system.

OBJECTIVE: To compare embryologic and clinical outcomes in terms of preimplantation development, implantation, pregnancy rates, and secretome profile of implanted blastocysts from the preimplantation genetic diagnosis program grown in sequential versus endometrial epithelial cell (EEC) coculture system. DESIGN: Retrospective clinical study and prospective experimental study. SETTING: In vitro fertilization clinical unit and university research laboratory. INTERVENTION(S): Blastomere biopsy, embryo culture, blastocyst transfer, and protein analysis of the media conditioned from implanted embryos obtained from coculture and sequential systems. MAIN OUTCOME MEASURE(S): Clinical study: blastocyst, implantation, and gestation rates in own and donated oocytes. Experimental study: differential protein analysis of implanted embryos grown in coculture system versus sequential system. RESULT(S): Of the 12,377 embryos analyzed, the blastocyst rates were 56.0% versus 45.9% in the coculture versus the sequential system, respectively, with own oocytes. With ovum donation, the rates were 70.5% versus 56.4%, respectively. Reproductive outcomes in terms of pregnancy rates (39.1% vs. 27.5%) and implantation rates (33.3% vs. 20.9%,) were statistically higher in EEC coculture versus sequential media. Furthermore, the protein profile of the EEC coculture versus the sequential system was obtained. Interleukin-6 (IL-6) was the most secreted protein by the EEC culture. Further ELISA experiments showed that the IL-6 present in the sequential medium diminished in implanted blastocysts. CONCLUSION(S): The coculture system favors blastocyst development and implantation rates, given the contribution of the factors secreted by endometrial epithelial cells, such as IL-6.

Impact of chromosomal abnormalities on preimplantation embryo development.

OBJECTIVES: To evaluate the influence of numerical chromosomal abnormalities on preimplantation embryo development. METHODS: This study includes 6936 embryos from 1245 women undergoing preimplantation genetic diagnosis (PGD). Indications for aneuploidy screening were: recurrent miscarriages, implantation failure, severe male factor, advanced maternal age, and mixed causes. Embryo biopsy was performed on day 3, and embryos were co-cultured until day 5, when embryo transfer was performed. RESULTS: In the aneuploidy screening regimen, normal euploid embryos showed significantly higher blastocyst rates (68.2%) compared to chromosomally abnormal (42.8%, p < 0.0001) and mosaic (53.7%, p < 0.0001) embryos. Among aneuploid embryos for autosomes, higher blastocyst rates were observed in trisomies than monosomies, although only statistically significant in patients over 36 years of age (50.8 vs 38.9%; p < 0.0001). In contrast, in embryos with sex chromosomes aneuploidy, similar blastocyst rates were observed between trisomies and monosomy X. CONCLUSION: Embryos with certain types of chromosomal abnormalities were negatively selected during preimplantation embryo development. Despite this selection, a remarkable percentage of chromosomally abnormal embryos can develop normally to blastocyst stage with high probability of implantation and pregnancy.

FISH screening of aneuploidies in preimplantation embryos to improve IVF outcome.

Preimplantation genetic diagnosis (PGD) has transformed the approach to the infertility patient in the IVF setting. Although the principal applications of PGD have been to prevent the transmission of sex-linked diseases, in time and with growing knowledge of the chromosomal abnormalities observed in preimplantation embryos, its applications have widened. Nowadays, apart from its implications in the prevention of transmission of chromosomal and genetic abnormalities, PGD is being used with increased frequency to improve the IVF outcome in patients with advanced maternal age (> or =38 years of age), recurrent miscarriage (> or =2 miscarriages), recurrent IVF failure (> or =3 failed IVF attempts) and severe male infertility. A high incidence of chromosomal abnormalities has been observed in these patient groups.

Clinical experience and perinatal outcome of blastocyst transfer after coculture of human embryos with human endometrial epithelial cells: a 5-year follow-up study.

OBJECTIVE: To evaluate the reproductive and neonatal outcome of blastocyst transfer after coculture with human endometrial epithelial cells in IVF and oocyte donation. DESIGN: Retrospective study. Private assisted reproductive center. PATIENTS(S): Two hundred sixty women undergoing IVF and 469 oocyte recipients. INTERVENTION(S): IVF or intracytoplasmic sperm injection (ICSI) and transfer of at least one blastocyst after coculture with human endometrial epithelial cells. MAIN OUTCOME MEASURE(S): Blastocyst formation rate, implantation and pregnancy rates, neonatal outcome, and congenital birth defects. RESULT(S): Among patients who had transfer with their own oocytes, 1193 of 2349 cocultured embryos developed up to the blastocyst stage (50.8%), and pregnancy and implantation rates of 33.9% and 19.2%, respectively, were achieved. In the oocyte donation program, 1819 blastocysts were obtained from 3127 embryos (58.2%), with subsequent pregnancy and implantation rates of 57.0% and 31.0%, respectively. The blastocyst rate remained stable throughout the 5 years of the study, but the pregnancy and implantation rates increased dramatically. Of 139 deliveries, 57 (41.0%) were multiple pregnancies and 1 (0.7%) was a multifetal birth (four live born infants). Out of 200 children born, 59% were male, and congenital birth defects were observed in 2.5%. CONCLUSION(S): Coculture of human embryos with endometrial epithelial cells yields a blastocyst formation rate of 50.8% to 58.2% and encouraging implantation and pregnancy rates. This technique reduces the mean number of embryos transferred in each patient. The number of embryos implanted is more relevant to neonatal outcome than is the coculture system and blastocyst transfer used. The risk of congenital birth defects associated with this program is similar to that recorded in early ET in IVF or ICSI.