Vitrification does not affect birth weight: lessons from the oocyte donation model.

RESEARCH QUESTION: Is embryo cryopreservation a cause of high birth weight and large for gestational age (LGA) in singletons resulting from vitrified-warmed embryo transfer? DESIGN: Retrospective cohort study evaluating 670 oocyte recipients who underwent fresh (367 cycles) or vitrified-warmed embryo transfer (303 cycles) at Instituto Bernabeu between July 2017 and March 2019. All single blastocyst transfers carried out in an artificial cycle that resulted in a singleton live birth were included. RESULTS: Maternal age (42.21 ± 4.45; 42.79 ± 3.83; P = 0.519), body mass index (23.34 ± 3.69; 23.80 ± 3.78; P = 0.075), gestational age (38.96 ± 1.97; 38.77 ± 2.15; P = 0.207), maternal smoking (10.8%; 13.0%; P = 0.475), gestational diabetes (4.9%; 4.3% P = 0.854), preeclampsia (2.7%; 5.6%; P = 0.074), hypertensive disorders (3.3%; 2.3%; P = 0.494), maternal parity (multiparous 18.5%; 14.5%; P = 0.177) and liveborn gender (female 44.5%; 48.8%; P = 0.276) were not significantly different between fresh or vitrified-warmed groups. Endometrial thickness was significantly higher in the fresh versus vitrified-warmed group (8.83 ± 1.73 versus 8.57 ± 1.59; P = 0.035, respectively). Oocyte donor height was similar between the fresh versus vitrified-warmed group (163.22 ± 5.88 versus 164.27 ± 6.66 cm; P = 0.057, respectively). Mean birth weight was not significantly different (3239.21 ± 550.43; 3224.56 ± 570.83; adjusted P = 0.058). No differences were observed in macrosomia (7.1%; 6.3%; adjusted OR 0.857, 95% CI 0.314 to 2.340, P = 0.764), LGA (6.0%; 6.7%; adjusted OR 0.450, 95% CI 0.176 to 1.149, P = 0.095), pre-term birth (10.9%; 9.0% adjusted P = 0.997), very pre-term birth (0.8%; 1.3%; adjusted P = 1.000), extremely pre-term birth (0%; 1.0%; adjusted P = 0.998); underweight (10.0%; 7.0%; adjusted P = 0.050); very low weight (0.6; 1.1%; adjusted P = 1.000) and small for gestational age (1.9%; 0.7%; adjusted P = 0.974) between fresh or vitrified-warmed groups. CONCLUSION: This study eliminates potential confounders that might influence fetal growth and demonstrates that embryo vitrification and warming procedures do not affect birth weight.

The Effect of Twisted Uterus Caused by Endometriosis or Myomatosis on Reproductive Treatment Outcomes.

Objective: Twisted uterus is detected when the body of the uterus is rotated from the cervical canal. This anomaly may be due to different causes, such as uterine fibroids, endometriosis or the presence of both. The study has aimed to compare the effect of the twisted uterus cause in terms of reproductive treatment outcomes. Materials and methods: It consisted of a retrospective study of twisted uterus cases with repeated implantation failure (more than three embryo transfers or four blastocysts transferred unsuccessfully) in our ultrasound department. The twisted uterus was defined when the vaginal probe needed to be rotated to assess the endometrial line thoroughly or when the coronal view was seen by 2D scan. From 2017 to 2020, 879 gynecological ultrasounds were performed. For statistical analysis, we carried out a logistical regression analysis adjusted by confounding factors. Results: From 145 patients included only 92 patients underwent reproductive treatments. With the known cause of uterine torsion. 56 patients with endometriosis, 18 with uterine myoma and the remaining 18 suffered from both. After assisted reproductive treatment, the endometriosis group showed the highest clinical pregnancy rate (53.57%) compared to myoma (22.22%) and endometriosis and myoma (38.89%) groups. Conclusion: Uterine myoma capable of causing uterine torsion may affect embryo implantation more than endometriosis. Prospective randomized studies with a larger number of patients would be needed to confirm these findings.

Methylenetetrahydrofolate reductase gene polymorphisms are not associated with embryo chromosomal abnormalities and IVF outcomes.

The aim of our study was to investigate the effect of maternal and embryo MTHFR C677T and A1298C polymorphisms on embryo aneuploidies and mosaicism and the correlation between these genetic variants in transferred euploid embryos and IVF outcomes. MTHFR genotype was analyzed in 77 women who performed an IVF/ICSI cycle with PGT-A. Moreover, to evaluate the effect of embryo MTHFR polymorphisms on embryo aneuploidies and mosaicism, the MTHFR genotype was analyzed in 191 biopsied embryos from the PGT-A cycles of these patients. Additionally, 218 DNA samples from trophectoderm biopsies belonging to a different group of patients were also genotyped. MTHFR polymorphisms were analyzed in a total amount of 409 trophectoderm samples. The main parameters analyzed were embryo aneuploidy and mosaicism rates. Finally, the IVF outcomes of 241 single euploid embryo transfers were assessed and compared between different MTHFR embryo genotypes. The aneuploidy rates were similar in embryos from homozygous normal women and women with at least one mutated allele (54.7% vs. 30.2% in 677C>T and 37.8% vs. 42.7% in 1298A>C). Furthermore, no differences were observed in the mosaicism rate (24.0% vs. 13.8% in 677C>T and 17.1% vs. 17.3% in 1298A>C). A similar analysis was performed, taking into account the embryo genotype results. No differences in aneuploidy rate were observed between the study groups. The only significant difference was the mosaicism rate among 677C>T genotype (13.5% in 677CC group vs. 5.4% in 677CT/TT; p = 0.019). Implantation rate, biochemical and clinical miscarriage rates, and ongoing pregnancy rate were compared between different embryo genotypes, and no statistically significant differences were found. In conclusion, the maternal MTHFR genotype did not influence embryo chromosomal abnormalities. Moreover, the embryo MTHFR genotype was not associated with embryo aneuploidy or IVF outcomes such as implantation, pregnancy loss, and ongoing pregnancy when euploid embryos were transferred.Abbreviations: MTHFR: methylenetetrahydrofolate reductase; IVF: in vitro fertilization; PGT-A: preimplantation genetic testing for aneuploidies; SAM: S-adenosyl methionine; SNP: single nucleotide polymorphism; SPSS: Statistical Package for Social Sciences; RIF: recurrent implantation failure; RPL: recurrent pregnancy loss; hCG: human chorionic gonadotropin; PBS: phosphate buffered saline; CGH: comparative genomic hybridization; NGS: next generation sequencing.

4D ultrasound as a method to assess uterine peristalsis.

OBJECTIVE: To study uterine peristalsis using step-by-step 4-dimensional (4D) ultrasound assessment video, explore its relationship with progesterone levels in a select in vitro fertilization population, and assess the reproducibility of the technique. DESIGN: Four-dimensional uterine ultrasound and a retrospective analysis of outcomes in relation with progesterone levels. The videos were also analyzed by a senior doctor, junior doctor, and a nurse for their reproducibility. SETTING: Instituto Bernabeu of Alicante is a private clinic. PATIENT(S): The study included 197 consecutive patients undergoing in vitro fertilization (from 2018 to 2019) with a history of recurrent implantation failure (defined as unsuccessful implantation of a total number of ≥3 blastocysts originated from oocyte donation cycles). Because it is known that most failures are attributed to the quality of the embryo, we deemed it important to explore the potential uterine factors explaining the failures in oocyte donation cycles, the use of which decreases the probability of embryo-related factors influencing it. INTERVENTION(S): The participants were evaluated for uterine contractions and serum progesterone levels (10-30 minutes before the embryo transfer procedure). Uterine contractility (UC) was assessed by recording a 6-minute-long video using a 4D mode (Voluson E10; General Electric, Boston, MA), which was performed by a single operator (B.M.). MAIN OUTCOMES MEASURE(S): The contractions were seen like waves going through the endometrial cavity. They were counted on a ×15 accelerated recording video. To define high-frequency UC, we separated uterine peristalsis (contractions per minute [cpm]) into quartiles. The highest quartile defined the hypercontractility group (>1.51 cpm; n = 41), considering the remaining quartiles as the normal contractility group (≤1.51 cpm; n = 156). The Mann-Whitney U test was performed. The intraclass correlation coefficient was used to validate variability. P <.05 was considered significant. SPSS version 21.0 was used for the statistical analysis. The institutional review board's approval was obtained. RESULT(S): Overall, an average of 1.1 cpm was found in the study population. There were no differences between the groups (hypercontractility vs. normal contractility) in terms of patient age and the presence of any uterine factor (adenomyosis, myomas, adhesions, or polyps). An inverse association was observed between UC and progesterone levels. Low progesterone levels (15.9 vs. 19.5 ng/mL; P = .027) were observed in the HUP and NUP group, respectively. The intraclass correlation coefficient to evaluate the interobserver variability was 0.75 (0.63-0.85; P = .000). CONCLUSION(S): Four-dimensional ultrasound assessment provides a dynamic view of uterine contractions, including their directionality and frequency. Even though recurrent implantation failure is yet a title of obscure definition and probably associated with multiple factors, a subgroup of patients with elevated UC associated with "low" progesterone levels may have a potential effect on their outcomes. Four-dimensional scan evaluation of UC constitutes a promising diagnostic tool in clinical practice; however, larger studies confirming our initial results are still pending.

Consistent results of non-invasive PGT-A of human embryos using two different techniques for chromosomal analysis.

RESEARCH QUESTION: Are discordances in non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) results attributable to the technique used for chromosomal analysis? DESIGN: A prospective blinded study was performed (September 2018 to December 2019). In total 302 chromosomal analyses were performed: 92 trophectoderm PGT-A biopsies and their corresponding spent embryo culture medium (SCM) evaluated by two methods (n = 184), negative controls (n = 8), and trophectoderm and inner cell mass biopsies from trophectoderm-aneuploid embryos (n = 18). Trophectoderm analyses were carried out using Veriseq (Illumina), and SCM was analysed using Veriseq and NICS (Yikon). RESULTS: Genetic results were obtained for 96.8% of trophectoderm samples versus 92.4% for both SCM techniques. The mosaicism rate was higher for SCM regardless of the technique used: 30.4% for SCM-NICS and 28.3% for SCM-Veriseq versus 14.1% for trophectoderm biopsies (P = 0.013, P = 0.031, respectively). No significant differences in diagnostic concordance were seen between the two SCM techniques (74.6% for SCM-NICS versus 72.3% for SCM-Veriseq; P = 0.861). For embryos biopsied on day 6, these rates reached 92.0% and 86.5%, respectively. On reanalysing trophectoderm-aneuploid embryos, the discrepancies were shown to be due to maternal DNA contamination (55.6%; 5/9), embryo mosaicism (22.2%; 2/9) and low resolution in SCM-NICS (11.1%; 1/9) and in both SCM techniques (11.1%; 1/9). CONCLUSIONS: This is the first study evaluating the consistency of different chromosomal analysis techniques for niPGT-A. In conclusion, the diagnostic concordance between PGT-A and niPGT-A seems independent of the technique used. Optimization of culture conditions and medium retrieval provides a potential target to improve the reliability of niPGT-A.

Monozygotic twinning following embryo biopsy at the blastocyst stage.

OBJECTIVE: Monozygotic twinning incidence following preimplantation genetic testing in embryos at cleavage-stage does not appear to increase; however, data regarding the possible impact of the blastocyst-stage preimplantation genetic testing is lacking. We compared the incidence of monozygotic twinning in preimplantation genetic testing cycles performed at the blastocyst-stage, versus cycles without PGT, following single embryo transfer. METHODS: In this retrospective cohort study, we analyzed the incidence of twin pregnancies in patients undergoing intracytoplasmic sperm injection and blastocyst-preimplantation genetic testing (253 cycles), versus a period-matched control population of patients undergoing intracytoplasmic sperm injection and single embryo transfer without preimplantation genetic testing (606 cycles). RESULTS: The overall monozygotic twinning rate was 14/859 (1.6%) per clinical pregnancy. The incidence of zygotic splitting following intracytoplasmic sperm injection and preimplantation genetic testing was 3.5% (95% Confidence interval 1.8%-6.6%) versus 0.8% (95% Confidence interval 0.3%-1.9%) following intracytoplasmic sperm injection without preimplantation sperm injection. After adjusting for potential confounders, preimplantation genetic testing cycles were associated with an increase in the incidence of monozygotic twinning when compared to cycles without embryo biopsy (Odd ratio 3.44, 95% Confidence interval 1.05-11.27, p=0.041). CONCLUSIONS: Our findings indicate that embryo biopsy for preimplantation genetic testing performed at the blastocyst stage is associated to an increase in the incidence of monozygotic twinning. Further validation in larger sample size studies is warranted. Patients undergoing preimplantation genetic testing must receive proper counselling about the potential risks of the technique.

Prevalence of candidate single nucleotide polymorphisms on p53, IL-11, IL-10, VEGF and APOE in patients with repeated implantation failure (RIF) and pregnancy loss (RPL).

Recurrent pregnancy loss (RPL; defined as the loss of three or more consecutive pregnancies) and recurrent implantation failure (RIF; when implantation is not achieved after at least three cycles of IVF) are two of the major challenges that reproductive medicine faces. Some polymorphisms have been identified as possible causes of an increased risk of these diseases. This paper studies the prevalence of the polymorphisms in p53, VEGF, IL-10, IL-11 and APOE in RIF and RPL patients that determines the risk for these pathologies. A total of 255 patients were selected (89 RPL patients, 77 RIF patients and 89 controls) and genotyped for p53-R72P; IL-11-1082-AG; VEGF-1154-AG; IL-10; APOE-R112C; APOE-R158C. Statistically significant differences were found in the prevalence of the E4 isoform (R122-R158) of the APOE gene in RPL patients (p < 0.05), and in RIF patients, the R72P polymorphism of the p53 gene and the 1154-AG of the VEGF gene showed different distribution (p < 0.05). Regarding the p53 and IL-11 studied polymorphisms, PP of p53 gene and GG of IL-11 are more prevalent in RPL patients without reaching statistical significance. In conclusion, our results suggest patients carrying variants in p53 and VEGF would be at risk of RIF, and those carrying variants in APOE gene would suffer RPL.

Ovarian hyperstimulation syndrome following GnRH agonist trigger for final follicular maturation in a patient undergoing random start ovarian stimulation for egg-donation cycle with an inadvertent concomitant early pregnancy.

We report the first case of OHSS following GnRH agonist trigger for final follicular maturation in random start ovarian stimulation for egg-donation cycles during inadvertent concomitant early pregnancy. As an additional note, the sustained activity exerted by the increasing endogenous hCG production seemed to be responsible for the suboptimal performance in terms of oocyte yield in the current case. OHSS can occur in random-start stimulations protocols even after the use of a GnRH agonist for triggering in case of concomitant unnoticed early pregnancy especially if stimulation is commenced in the periovulatory/luteal phase. The present case report introduces a note of extreme caution when proceeding with this protocol in an otherwise fertile population (egg-donors, elective or oncologic oocyte cryopreservation).

Second-generation preimplantation genetic testing for aneuploidy in assisted reproduction: a SWOT analysis.

Second-generation preimplantation genetic testing for aneuploidy (PGT-A 2.0) in patients with an unfavourable reproductive and IVF prognosis is becoming common practice, with the aim of improving reproductive outcomes. However, there is still no clear evidence on the possible advantages and drawbacks with regard to this procedure. In this discussion paper, based on a SWOT (strengths, weaknesses, opportunities, threats) analysis, the different aspects of this strategy are evaluated. Current evidence suggests that PGT-A 2.0 should not at present have an indiscriminate application, but it might be indicated in cases in which the risk of aneuploidy is increased.

The relevance of the individual screening for genetic variants in predicting ovarian response.

OBJECTIVE: To investigate if polymorphisms of some genes involved in folliculogenesis predict ovarian response. METHODS: This prospective randomized study includes 124 egg donors genotyped for six SNPs ESR1 (rs2234693), AMHR2 (rs2002555), GDF-9 (rs10491279 and rs254286), AMH (rs10407022) and LHCBR (rs229327) genes and four STRs in ESR1 rs3138774), SHBG (rs6761), CYP19A1 (rs60271534) and AR genes (CAG repeats in exon 1). All donors followed standard ovarian stimulation protocol using a daily dose of 225 UI. The genotypes obtained were compared with the ovarian stimulation outcome. RESULTS: Regarding the number of retrieved oocytes, we found statistical differences for the ESR1 SNP and STR (19.3 ± 8.9 for TT vs 15.3 ± 6.2 for CC/CT, P = 0.027; 19.1 ± 8.3 for <17repeats vs 14.7 ± 6.2 for >17repeats, P = 0.020). Moreover, women carrying TT in the ESR1 at position c.-397T>C with ESR1 (TA)n=17 retrieved the highest number of oocytes (20.4 ± 9.3) (P = 0.001). Concerning AMHR2, we observed an association with the length of stimulation (9.1 ± 1.4 d for AA vs 9.7 ± 1.3 d for AG/GG, P = 0.021) and gonadotropin received (2050 ± 319 for AA vs 2188 ± 299 for AG/GG, P = 0.017). No significant differences were observed for the other polymorphisms (P > 0.05). CONCLUSION: The polymorphisms in ESR1 and AMHR2 genes showed a clear association with the number of retrieved oocytes and the stimulation data, respectively. Our results suggest that polymorphisms in the genes for key reproductive hormones receptors could be used to predict the ovarian response and to personalize the stimulation prior the treatment.

Androgen receptor CAG repeat length is associated with ovarian reserve but not with ovarian response.

The human androgen receptor (AR) gene contains a highly polymorphic CAG repeat sequence within exon 1. In-vitro studies have shown a relationship between CAG repeats in the AR gene and its transactivation potential. This variation in length may play a role in anovulatory infertility. The objective of this study was to investigate whether CAG polymorphism of the AR gene has a predictive value for ovarian reserve, response and cycle outcome in an egg donor programme. CAG length of the AR gene was determined in 147 oocyte donors. All donors underwent ovarian stimulation with a gonadotrophin-releasing hormone antagonist protocol (n = 355). No differences were reported in days of stimulation, gonadotrophin doses, and number of oocytes retrieved. Clinical outcomes were not affected by the CAG repeat length of the AR gene; the primary end-point, antral follicle count, was significantly affected (P < 0.05). In conclusion, in a population of fertile egg donors AR gene CAG polymorphism does not affect ovarian response to gonadotrophins. Antral follicle count was associated with the CAG polymorphism genotype. This suggests that genetic factors may increase susceptibility to poor ovarian reserve, and that AR gene genotype could play a role in the natural ovarian ageing process.

Pharmacogenetics of ovarian response.

Effective controlled ovarian stimulation (COS) is crucial for IVF outcome. Ovarian response to follicle-stimulating hormone, however, varies widely among women undergoing ovarian stimulation. Advance identification of patients who will elicit a poor or high response to standard treatment would be of great clinical benefit for such patients. Application of pharmacogenetics to ovarian response may predict stimulation success but also help in the adjustment and design of doses prior to treatment. Different studies have examined the impact of variations in follicle-stimulating hormone receptor, biochemical pathways involved in estrogen production and action, folliculogenesis and other aspects. Recently, gene-association studies have tried to identify a number of genetic variations affecting interindividual variability in COS.

Negative effect of P72 polymorphism on p53 gene in IVF outcome in patients with repeated implantation failure and pregnancy loss.

PURPOSE: Investigate whether R72P on p53 gene polymorphism has a higher prevalence among women with a history of recurrent implantation failure (RIF) and pregnancy loss (RPL) and its influence in their IVF cycle outcome. MATERIAL AND METHODS: p53 polymorphism R72P has been studied in 181 women. The control group included 83 oocyte donors. In the study group 98 women were included: 44 with RIF and 54 with RPL. From the study group, 76 patients underwent IVF-cycles (55 RPL and 21 RIF). RESULTS: The frequency of PP genotypes on p53 among RIF was 11.4% compared with 18.5% for RPL and 6% in controls (p < 0.01). There were no significant differences with respect to patient characteristics. Significant differences were reported in pregnancy rate (69.4% for RR/RP and 33.3% for PP; p < 0.05), embryo implantation rate (33.3% for RR/RP and 7.3% for PP; p < 0.05) and ongoing pregnancy rate (53.1% for RR/RP and 14.3% for PP; p < 0.05) among RIF and RPL. CONCLUSIONS: This investigation reveals that in RIF and RPL patients R72P on p53 gene is more prevalent than fertile population. Moreover, patients carrying a PP genotype on p53 codon 72 will have less chance to achieve an ongoing pregnancy. This information together with some additional markers will allow development of diagnostic tests for detects risk for RIF and RPL before infertility treatment is initiated.

Effect of follicle-stimulating hormone receptor N680S polymorphism on the efficacy of follicle-stimulating hormone stimulation on donor ovarian response.

OBJECTIVE: The aim of this study was to investigate whether N680S FSHR polymorphism has a predictive value for the ovarian response to stimulation with gonadotropins and cycle outcome in our egg donor program. METHODS: The oocyte donor candidates were selected according to the Instituto Bernabeu egg donation program requirements and ASRM and ESHRE guidelines for oocyte donation. The FSHR polymorphism N680S was studied in 145 oocyte donors. All donors underwent controlled ovarian hyperstimulation (COH) (n=355) using urinary follicle-stimulating hormone in a GnRH antagonist protocol and receiving a GnRH agonist triggering. The main outcome measures were oocyte yield, days of stimulation, gonadotropin doses, biochemical pregnancy, ongoing pregnancy, and miscarriage rates. RESULTS: Significant differences were reported in the antral follicle count (16.5 ± 5.0 for NN, 14.5 ± 4.7 for NS, and 14.1 ± 3.8 for SS), number of eggs retrieved (21.5 ± 9.2 for NN, 18.5 ± 8.2 for NS, and 19.8 ± 8.9 for SS), and gonadotropin doses (2098.5 ± 639.4 IU for NN, 2023 ± 490.1 IU for NS, and 2149.5 ± 552.3 IU for SS) between the genotypes. The clinical outcome was not affected by the N680S polymorphism of the FSHR gene in the egg donors. CONCLUSION: In a population of fertile egg donors, the FSHR gene polymorphism at position 680 is associated with different ovarian responses to COH. The genotype of the FSHR gene is an important factor for determining the prognosis of the COH cycles in normo-ovulatory fertile women.

Preimplantation genetic diagnosis of X-linked retinoschisis.

The aim of this study was to perform preimplantation genetic diagnosis (PGD) for X-linked retinoschisis using multiple displacement amplification (MDA) for whole genome amplification and linked markers to the RS1 gene. The study evaluates the ability of MDA to amplify the whole genome directly from a single blastomere. MDA products were used for polymerase chain reaction analysis of two polymorphic markers flanking the RS1 gene and a new X/Y marker, X22, to sex embryos in an X-linked retinoschisis PGD programme. Two couples in whom the wives were carriers of the RS1 gene mutation (599G-->A), previously identified in their families, were subjected to two PGD cycles each. The main outcome measure was the ability to analyse single blastomeres for X-linked retinoschisis using MDA. As a result, the development of an MDA-PGD protocol for X-linked retinoschisis allowed for the diagnosis of 20 embryos in the four PGD cycles performed. These were biopsied on day 3 of culture and analysed. Eight embryos were affected males and two embryos were female carriers. In summary, three healthy female and four healthy male embryos, and a female carrier embryo, were transferred 48 h after biopsy. One single pregnancy was achieved. This report shows that the MDA technique is useful for overcoming the problem of insufficient genomic DNA in PGD. It also allows the simultaneous amplification of different targets to perform diagnosis of any known gene defect and sexing test by standard methods and conditions.