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Di-(2-ethylhexyl) phthalate (DEHP) and Cadmium (Cd) affect female reproduction. To date, toxicological research has focused on the effects of individual contaminants, whereas living beings are exposed to mixtures. This study analyzed the effects of a DEHP/Cd mixture on nuclear and cytoplasmic maturation of sheep cumulus–oocyte complexes (COCs) compared with single compounds. COCs recovered from slaughterhouses-derived sheep ovaries were in vitro exposed to 0.5 μM DEHP, 0.1 μM Cd, or DEHP/Cd mixture at the same concentrations during 24 h of in vitro maturation (IVM). After IVM, oocyte nuclear chromatin configuration was evaluated, and bioenergetic/oxidative parameters were assessed on expanded cumulus cells (CCs) and matured oocytes (chi-square test and one-way ANOVA; p < 0.05). Under examined conditions, oocyte nuclear maturation was never impaired. However, COC bioenergetics was affected with stronger effects for the mixture than single compounds. Indeed, the percentages of matured oocytes with healthy mitochondrial distribution patterns were reduced (p < 0.001 and p < 0.05 for mixture and single compounds, respectively). Oocyte mitochondrial membrane potential, intracellular ROS levels, and mitochondria/ROS co-localization were reduced, with the same significance level, in all contaminated conditions. CCs displayed increased ROS levels only upon mixture exposure (p < 0.001). In conclusion, in vitro exposure to the DEHP/Cd mixture affected COC quality in the sheep to a greater extent than separate compounds. [ABSTRACT FROM AUTHOR]

Follitropin delta (Δ) is a recombinant human follicle-stimulating hormone (rFSH), like natural human FSH, that can stimulate the development and growth of multiple follicles. Treatment with Follitropin-Δ may cause mild to severe adverse reactions, such as the risk of developing ovarian hyperstimulation syndrome, resulting in nausea, vomiting and diarrhea, weight loss, respiratory difficulty, stomach swelling and discomfort of the pelvic area, headaches, and fatigue. To date, the effects of a Follitropin-Δ overdosage are unknown, and no data are reported in the scientific literature or in the drug data sheet. Therefore, this study aimed to describe the effects of Follitropin-Δ overdosages in poorly responding women who underwent IVF cycles. This is a descriptive case series of four nulligravid, poorly responding patients, two of whom made requests for fertility preservation. Four poorly responding patients who were prescribed 20.0 µg/day of Follitropin-Δ for three consecutive days wrongly injected the total cartridge of 72 µg Follitropin-Δ every day. After the incorrect injection of Follitropin-Δ, the patients continued their controlled ovarian stimulation and underwent vaginal ovarian pick up. The analyzed patients had no side effects or adverse reactions. The evaluations reported in this case series showed that the accidental use of 72 µg/day of Follitropin-Δ for three days did not cause side effects or adverse reactions in poor responders. [ABSTRACT FROM AUTHOR]

Simple Summary: In recent years, due to industry and market preferences, local animal breeds have been exposed to genetic erosion and extinction risk. Effective strategies for their recovery and conservation are needed. Gentile di Puglia (GdP) is an autochthonous sheep breed, typical of Southern Italy, with an aptitude for wool, meat, and milk production and considerable historical and cultural value. The development of a GdP gamete and embryo cryobank could help to support the numerical reimplementation of this population. Animal germplasm conservation is mainly performed through sperm cryopreservation, whereas strategies on the female side are developed in a limited way. In this study, the dual purpose of monitoring the reproductive efficiency in one pilot GdP farm in the Apulia region and setting up a cryopreservation protocol, by vitrification, of immature cumulus-oocyte complexes (COCs) recovered from pre-pubertal lambs, followed by an analysis of their in vitro maturation potential and bioenergetic-oxidative status, was pursued. The results indicated that traditional reproductive management leads to progressive offspring reduction and that ex situ biotechnological conservation strategies, through immature oocyte vitrification and in vitro maturation, can support in situ conservation, leading to in vitro embryo production and transfer. Gentile di Puglia (GdP) is an autochthonous sheep breed of Southern Italy included among ovine breeds threatened by genetic erosion and extinction risk, which have been given attention by local and international institutions, thus emphasizing the need for germplasm conservation actions. In the present study, two assisted reproduction approaches, finalized for GdP conservation, were performed: (1) on-farm reproductive efficiency evaluation, expressed as pregnancy rate (PR), twin pregnancy rate (tPR), and body condition score (BCS), for three consecutive breeding cycles and (2) pre-pubertal lambs' immature cumulus–oocyte complex (COC) retrieval, vitrification, in vitro maturation (IVM), and assessment of meiotic stage and bioenergetic-oxidative status compared with those of other Italian and European commercial breeds. PR and tPR were progressively reduced over time. In all clinical examination times, BCS was significantly lower in nonpregnant ewes compared with pregnant ones. Fresh GdP pre-pubertal lamb COCs achieved meiotic maturation and showed healthy bioenergetic–oxidative status after IVM. Vitrification reduced the oocyte maturation rate in all groups. However, mature oocytes retained their cytoplasmic maturity, expressed as a mitochondria distribution pattern and activity, indicating promising developmental competence. In conclusion, clinical- and biotechnological-assisted reproduction approaches can support conservation strategies of GdP and other local sheep breeds in Southern Italy. [ABSTRACT FROM AUTHOR]

Simple Summary: Cadmium (Cd), a highly toxic environmental contaminant, negatively affects human and animal fertility in females. In sheep, Cd displays age-dependent bioaccumulation at ovarian level. At environmental nanomolar concentrations, it reduces oocyte fertilization by inducing oxidative stress on cumulus cells (CCs), a cell population of the cumulus-oocyte complex (COC) supporting oocyte growth, functional maturation and fertilization. In this study, the modifications induced by Cd exposure to all the genes expressed in CCs of in vitro matured COCs, recovered from the ovaries of adult and prepubertal sheep, were analysed by RNA sequencing. A set of genes significantly dysregulated upon Cd exposure was identified. Effects of Cd were more relevant in CCs from adult than from prepubertal COCs. Most genes were upregulated while a minority of them were downregulated. Some genes were already known as involved in ovarian activity or Cd-induced effects, whereas others were completely new in these fields. These findings identify in the sheep, an important livestock species with translational relevance in human reproduction, the set of genes controlling oocyte functional competence, altered by Cd. These biomarkers will make it possible to identify oocytes that cannot be fertilized to evaluate whether they are to be discarded or recovered with detoxifying treatments. Cadmium (Cd), a highly toxic pollutant, impairs oocyte fertilization, through oxidative damage on cumulus cells (CCs). This study analysed the transcriptomic profile of CCs of cumulus-oocyte complexes (COCs) from adult and prepubertal sheep, exposed to Cd nanomolar concentration during in vitro maturation. In both age-groups, CCs of matured oocytes underwent RNA-seq, data analysis and validation. Differentially expressed genes (DEGs) were identified in adult (n = 99 DEGs) and prepubertal (n = 18 DEGs) CCs upon Cd exposure. Transcriptomes of adult CCs clustered separately between Cd-exposed and control samples, whereas prepubertal ones did not as observed by Principal Component Analysis. The transcriptomic signature of Cd-induced CC toxicity was identified by gene annotation and literature search. Genes associated with previous studies on ovarian functions and/or Cd effects were confirmed and new genes were identified, thus implementing the knowledge on their involvement in such processes. Enrichment and validation analysis showed that, in adult CCs, Cd acted as endocrine disruptor on DEGs involved in hormone biosynthesis, cumulus expansion, regulation of cell signalling, growth and differentiation and oocyte maturation, whereas in prepubertal CCs, Cd affected DEGs involved in CC development and viability and CC-oocyte communications. In conclusion, these DEGs could be used as valuable non-invasive biomarkers for oocyte competence. [ABSTRACT FROM AUTHOR]

In conventional assisted reproductive technologies (ARTs), oocytes are in vitro cultured in static conditions. Instead, dynamic systems could better mimic the physiological in vivo environment. In this study, a millifluidic in vitro oocyte maturation (mIVM) system, in a transparent bioreactor integrated with 3D printed supports, was investigated and modeled thanks to computational fluid dynamic (CFD) and oxygen convection-reaction-diffusion (CRD) models. Cumulus-oocyte complexes (COCs) from slaughtered lambs were cultured for 24 h under static (controls) or dynamic IVM in absence (native) or presence of 3D-printed devices with different shapes and assembly modes, with/without alginate filling. Nuclear chromatin configuration, mitochondria distribution patterns, and activity of in vitro matured oocytes were assessed. The native dynamic mIVM significantly reduced the maturation rate compared to the static group (p < 0.001) and metaphase II (MII) oocytes showed impaired mitochondria distribution (p < 0.05) and activity (p < 0.001). When COCs were included in a combination of concave+ring support, particularly with alginate filling, oocyte maturation and mitochondria pattern were preserved, and bioenergetic/oxidative status was improved (p < 0.05) compared to controls. Results were supported by computational models demonstrating that, in mIVM in biocompatible inserts, COCs were protected from shear stresses while ensuring physiological oxygen diffusion replicating the one occurring in vivo from capillaries. [ABSTRACT FROM AUTHOR]

Diagnostic imaging has significantly grown over the last thirty years as indispensable support for diagnostic, prognostic, therapeutic and monitoring procedures of human diseases. This study explored the effects of low-dose X-ray medical diagnostics exposure on female fertility. To aim this, cumulus-oocyte complexes (COCs) recovered from the ovaries of juvenile sheep and human ovaries were used as complementary models for in vitro studies. In the sheep model, the effects of low-dose X-rays on oocyte viability and developmental competence were evaluated. In human ovaries originated from two age group (21–25 and 33–36 years old) subjects with gender dysphoria, X-rays effects on tissue morphology, follicular density and expression of apoptosis-related (NOXA, PUMA, Bcl2, Bak, γH2AX) and cell cycle-related genes (p21 and ki67) were investigated. It was noted that in sheep, the minimum dose of 10 mGy did not influence most of examined parameters at oocyte and embryo levels, whereas 50 and 100 mGy X-ray exposure reduced oocyte bioenergetic/oxidative activity but without any visible effects on oocyte and embryo development. In addition, blastocyst bioenergetic/oxidative status was reduced with all used doses. Overall data on human ovaries showed that low-dose X-rays, similarly as in sheep, did not alter any of examined parameters. However, in women belonging to the 33–36 year group, significantly reduced follicular density was observed after exposure to 50 and 100 mGy, and increased NOXA and Bax expression after exposure at 50 mGy. In conclusion, used low-doses of X-ray exposure, which resemble doses used in medical diagnostics, produce weak damaging effects on female fertility with increased susceptibility in advanced age. [ABSTRACT FROM AUTHOR]

The genotoxic and nephrotoxic mycotoxin Ochratoxin A (OTA) has also been reported to have adverse effects on oocyte maturation and embryo development. Previous studies on the effects of OTA on female fertility have used micromolar concentrations, but no information is available to date on effects in a more relevant nanomolar range. This study used a juvenile sheep model to evaluate the effects of oocyte exposure to low levels of OTA on maturation, fertilization, and embryo development. Further, it was investigated whether different mechanisms of action of OTA could be responsible for varying toxic effects at different levels of exposure. Cumulus-oocyte-complexes (COCs) were exposed to 10 μmol/L–0.1 nmol/L OTA during in vitro maturation and evaluated for cumulus viability, oocyte maturation, and bioenergetic/oxidative status. COCs were subjected to in vitro fertilization, embryo culture, and embryo quality assessment via morphology, viability, bioenergetic/oxidative status, and time-lapse monitoring. At micromolar concentrations, OTA induced cytotoxic effects, by reducing cumulus expansion and oocyte maturation. OTA altered temporospatial dynamics of zygote pronuclear formation and embryo morphokinetics. Blastocysts, even morphologically normal, were found to undergo collapse events, which were probably related to boosted blastocyst mitochondrial activity. At nanomolar concentrations, OTA did not affect COC morpho-functional parameters, but impaired oocyte ability to prevent polyspermy and increased blastocyst apoptosis. In conclusion, in the female germ cell, cytotoxic nonspecific effects characterize OTA-induced toxicity at high exposure levels, whereas fine tuning-mode effects, not associated with altered cell viability and integrity, characterize OTA toxic action at low levels. [ABSTRACT FROM AUTHOR]

Three-dimensional in vitro maturation (3D IVM) is a promising approach to improve IVM efficiency as it could prevent cumulus-oocyte complex (COC) flattening and preserve its structural and functional integrity. Methods reported to date have low reproducibility and validation studies are limited. In this study, a bioprinting based production process for generating microbeads containing a COC (COC-microbeads) was optimized and its validity tested in a large animal model (sheep). Alginate microbeads were produced and characterized for size, shape and stability under culture conditions. COC encapsulation had high efficiency and reproducibility and cumulus integrity was preserved. COC-microbeads underwent IVM, with COCs cultured in standard 2D IVM as controls. After IVM, oocytes were analyzed for nuclear chromatin configuration, bioenergetic/oxidative status and transcriptional activity of genes biomarker of mitochondrial activity (TFAM, ATP6, ATP8) and oocyte developmental competence (KHDC3, NLRP5, OOEP and TLE6). The 3D system supported oocyte nuclear maturation more efficiently than the 2D control (P<0.05). Ooplasmic mitochondrial activity and reactive oxygen species (ROS) generation ability were increased (P<0.05). Up-regulation of TFAM, ATP6 and ATP8 and down-regulation of KHDC3, NLRP5 expression were observed in 3D IVM. In conclusion, the new bioprinting method for producing COC-microbeads has high reproducibility and efficiency. Moreover, 3D IVM improves oocyte nuclear maturation and relevant parameters of oocyte cytoplasmic maturation and could be used for clinical and toxicological applications. [ABSTRACT FROM AUTHOR]

Juvenile in vitro embryo technology (JIVET) provides exciting opportunities in animal reproduction by reducing the generation intervals. Prepubertal oocytes are also relevant models for studies on oncofertility. However, current JIVET efficiency is still unpredictable, and further improvements are needed in order for it to be used on a large-scale level. This study applied bioengineering approaches to recreate: (1) the three-dimensional (3D) structure of the cumulus–oocyte complex (COC), by constructing—via bioprinting technologies—alginate-based microbeads (COC-microbeads) for 3D in vitro maturation (3D-IVM); (2) dynamic IVM conditions, by culturing the COC in a millifluidic bioreactor; and (3) an artificial follicular wall with basal membrane, by adding granulosa cells (GCs) and type I collagen (CI) during bioprinting. The results show that oocyte nuclear and cytoplasmic maturation, as well as blastocyst quality, were improved after 3D-IVM compared to 2D controls. The dynamic 3D-IVM did not enhance oocyte maturation, but it improved oocyte bioenergetics compared with static 3D-IVM. The computational model showed higher oxygen levels in the bioreactor with respect to the static well. Microbead enrichment with GCs and CI improved oocyte maturation and bioenergetics. In conclusion, this study demonstrated that bioengineering approaches that mimic the physiological follicle structure could be valuable tools to improve IVM and JIVET. [ABSTRACT FROM AUTHOR]