Type of protein supplement in cryopreservation solutions impacts on the degree of ultrastructural damage in frozen-thawed human oocytes

Abstract Protein sources used as supplements of IVF culture media are known to have several implications for the function and stability of embryo culture environment. In fact, they i) transport biologically active molecules ii) chelate heavy metals, iii) regulate media pH, iii) scavenge reactive oxygen species (ROS) and iv) attenuate osmotic stress to which cells are exposed in sub-optimal culture conditions. Instead, their specific relevance to the formulation of cryopreservation solutions used for gamete and embryo cryopreservation remains uncertain. In the present work, we tested the hypothesis that different protein supplements present in cryopreservation solutions, serum or plasma protein solution (PPS), or different concentrations of the same supplement (serum), are associated with different types and/or magnitude of cryopreservation-derived cell damage. To this end, using cryopreservation solutions containing serum or PPS, donated supernumerary human mature oocytes were frozen-thawed by slow freezing and compared with fresh controls. Ultrastructural markers of oocyte quality were adopted as objective measure to assess possible damage from cryopreservation. The study results indicate that the adoption of serum minimises cell damage induced by cryopreservation. Indeed, typical hallmarks of cryodamage in human oocytes, i.e. loss of cortical granules, zona pellucida hardening and above all vacuolization, were largely reduced in oocytes cryopreserved with solutions containing serum, especially if used a higher concentration. This suggest that oocyte cryopreservation still has significant margins of improvement that may derive also from composition of cryopreservation media

The Impact of Unbalanced Maternal Nutritional Intakes on Oocyte Mitochondrial Activity: Implications for Reproductive Function.

Abstract Accumulating evidence on the effect of nutrition on reproduction is emerging from both animal and human studies. A healthy dietary pattern and nutrient supplementation, especially during the peri-conceptional period, might be helpful to achieve a live birth, although the mechanisms implicated are not fully understood. The endocrine system and the ooplasmic organelles apparatus, in particular the mitochondria, are clearly key elements during oogenesis and subsequent embryo development, and their proper functioning is associated with nutrition, even beyond maternal aging. Several studies in animal models have reported various adverse effects on mitochondria caused by unbalanced dietary intakes such as high fat diet, high fat high sugar diet, and low protein diet. The alterations produced might include mitochondrial intracellular distribution, content, structure, biogenesis, and functioning. This review summarizes the key role of mitochondria in female reproduction and the effects of different dietary macronutrient compositions on oocyte mitochondrial activity with their possible short-, medium-, and long-term effects

Ultrastructural assessment of human metaphase II oocytes after cryopreservation with media containing different macromolecular supplements

Study question: To verify whether type and concentration of protein supplement included in freezing solutions affect the ultrastructure of human metaphase II (MII) oocytes cryopreserved by slow cooling and therefore optimize cryopreservation conditions. Summary answer: This study confi rms: (1) slow freezing ensures good preservation of the oocyte; (2) premature cortical granules (CG) exocytosis and vacuolization are both markers of cryodamage; (3) prolonged culture before cryopreservation may cause enlargement of mitochondria-vesicle (MV) complexes. Furthermore serum supplementation induces good preservation of the ooplasm avoiding extensive vacuolization. What is known already: Cryoprotective agents (CPAs) are essential components in freezing solutions, but may also disrupt the meiotic spindle and organelles. Together with conventional CPAs, protein supplement is known to preserve cell structure. CG exocytosis requires a healthy plasma membrane and cytoskeleton. This process may be affected by cryopreservation as a result of shrinkage during CPA addition leading to subjacent localization of cortical granules and resulting in release of their contents because of plasma membrane fusion after rewarming. Study design, size, duration: Forty supernumerary MII oocytes were donated by consenting patients (aged 28–36) enrolled in an IVF program. Thirty-four oocytes were cryopreserved using slow freezing with 0.2M sucrose, 1.5M 1–2 propanediol (PROH) and either serum or Plasma Protein Solution (PPS) in the freezing mixture. Six oocytes were used as fresh controls. Participants/materials, setting, methods: Oocytes were cryopreserved with 20% (n = 12) and 10% (n = 10) serum or 10% PPS (n = 12). Samples were fi xed by 2 h after thawing and prepared for light and transmission electron microscopy (LM and TEM) for ultrastructural analysis of CG, mitochondria-smooth endoplasmic reticulum aggregates (M-SER) and vacuolization. Main results and the role of chance: By LM, both control and cryopreserved oocytes appeared rounded and with uniform distribution of organelles. By TEM, M-SER and small (MV) complexes were the most numerous structures found in all oocytes. Only in a few cryopreserved oocytes, irrespective of macromolecular supplement, numerous large MV complexes were found, probably due prolonged culture (3–4 h) before cryopreservation. Amount and density of CG appeared abnormally reduced in all samples. Different degrees of vacuolization were present in the ooplasm of cryopreserved, but not fresh oocytes. Extensive vacuolization was present only in a minority of oocytes cryopreserved with serum (16.6% of the oocytes supplemented with 20% serum and 20% of the oocytes supplemented with 10% serum), whereas a higher number (66.6%) of oocytes supplemented with 10% PPS were largely vacuolized. Limitations, reason for caution: Although the interest raised by the investigation of which macromolecular supplement gives best results in terms of ultrastructural preservation of oocyte quality and integrity, this study should be extended in order to verify this fi nding in clinical routine. Wider implications of the fi ndings: This approach can be considered of interest for different cryopreservation methods extensively adopted in assisted reproductive treatments. In fact, the matter of protein supplement in different formulations and concentrations is still debated both for culture media supplementation and vitrification solutions

TERRA: A novel biomarker of embryo quality and art outcome

none10noTelomeres are considered to be an internal biological clock, and their progressive shortening has been associated with the risk of age-related diseases and reproductive alterations. Over recent years, an increasing number of studies have focused on the association between telomere length and fertility, identifying sperm telomere length (STL) as a novel biomarker of male fertility. Although typically considered to be repeated DNA sequences, telomeres have recently been shown to also include a long non-coding RNA (lncRNA) known as TERRA (telomeric repeat-containing RNAs). Interestingly, males with idiopathic infertility show reduced testicular TERRA expression, suggesting a link between TERRA and male fertility. The aim of this study was to investigate the role of seminal TERRA expression in embryo quality. To this end, STL and TERRA expression were quantified by Real Time qPCR in the semen of 35 men who underwent assisted reproductive technologies (ART) and 30 fertile men. We found that TERRA expression in semen and STL was reduced in patients that underwent ART (both p < 0.001). Interestingly, TERRA and STL expressions were positively correlated (p = 0.010), and TERRA expression was positively associated with embryo quality (p < 0.001). These preliminary findings suggest a role for TERRA in the maintenance of sperm telomere integrity during gametogenesis, and for the first time, TERRA expression was found as a predictive factor for embryo quality in the setting of assisted reproduction.noneRocca M.S.; Dusi L.; Di Nisio A.; Alviggi E.; Iussig B.; Bertelle S.; De Toni L.; Garolla A.; Foresta C.; Ferlin A.Rocca, M. S.; Dusi, L.; Di Nisio, A.; Alviggi, E.; Iussig, B.; Bertelle, S.; De Toni, L.; Garolla, A.; Foresta, C.; Ferlin, A

Endometriosis shows no impact on the euploid blastocyst rate per cohort of inseminated metaphase-II oocytes: A case-control study

Objective: To evaluate the true impact of endometriosis on oocytes’ competence defined as blastulation, euploidy and implantation rates. Design: Retrospective multicenter case-control study involving infertile couples undergoing ICSI with qPCR and trophectoderm biopsy-based PGT-A. Patients affected from endometriosis (n = 210) were diagnosed through transvaginal sonography or surgical history with histological confirmation. Each case was matched to two controls (n = 420) according to IVF clinic, maternal age at retrieval (38.6 ± 2.7 yr), number of previous failed IVF treatments (0.5 ± 0.8) and number of metaphase-II oocytes retrieved (6.1 ± 3.7 per patient). The primary outcome was the mean euploid blastocyst rate per cohort of inseminated metaphase-II oocytes. Other embryological, clinical, obstetric and neonatal outcomes were also evaluated. Results: The mean euploid blastocyst rate per cohort of inseminated metaphase-II oocytes was identical in the two groups (18 %±22 %) independently of maternal age. No difference was shown for all embryological outcomes investigated. The live birth rates per vitrified-warmed single euploid blastocyst transfer were also similar (67/158, 42 % in patients affected from endometriosis versus 132/327, 40 % in matched-controls). No difference was reported in the gestational and neonatal outcomes. The cumulative live birth delivery rates among completed cycles were also identical (61/201, 30 % versus 117/391, 30 % in endometriosis and matched-control groups, respectively) independently of maternal age. Conclusions: Endometriosis might not impair oocyte developmental and reproductive competence, although its potential impact on the number of metaphase-II oocytes retrieved cannot be ignored. This information is critical for clinicians during counseling to outline an effective strategy to treat infertile patients affected from this condition. Future prospective studies are needed to evaluate the impact of endometriosis stage on euploidy rates

Maternal body mass index associates with blastocyst euploidy and live birth rates: the tip of an iceberg?

Research question: Does maternal preconceptional body mass index (BMI) associate with mean blastocyst euploidy rate (m-ER) per patient and live birth rate (LBR) after vitrified-warmed euploid single embryo transfer (SET)? Design: Observational study conducted between April 2013 and March 2020 at a private IVF clinic, involving 1811 Caucasian women undergoing trophectoderm biopsy and comprehensive chromosome testing. The outcomes of 1125 first vitrified-warmed euploid SET were also analysed. Patients were clustered as normal weight (BMI 18.5-25; n = 1392 performing 859 SET), underweight (BMI 25; n = 259 performing 154 SET). m-ER per patient was the primary outcome. The secondary outcomes were all clinical outcomes per euploid SET. All data were adjusted for confounders through regression analyses. Results: The m-ER per patient decreases as maternal BMI increases from 17 up to 22-23 before reaching a plateau. A linear regression adjusted for maternal age confirmed this moderate association (unstandardized coefficient B: -0.6%, 95% confidence interval [CI]: -1.1 to -0.1%, P = 0.02). All clinical outcomes were similar between normal weight and underweight women. Overweight women, instead, showed higher miscarriage rate per clinical pregnancy (n = 20/75, 26.7% versus n = 67/461, 14.5%; odds ratio [OR] adjusted for blastocyst quality and day of full blastulation: 2.0, 95% CI: 1.1-3.6, P = 0.01) and lower LBR per SET (n = 55/154, 35.7% versus n = 388/859, 45.2%; OR adjusted for blastocyst quality and day of full blastulation: 0.67, 95% CI: 0.46-0.96, P = 0.03). Conclusion: These data indicate a need for future research on more sensitive metrics to assess body fat mass and distribution, as well as on the mechanisms leading to lipotoxicity, thereby impairing embryo competence and/or endometrial receptivity. Overweight women should be informed of their higher risk for miscarriage and, whenever possible, encouraged to lose weight, especially before transfer