During human in vitro culture, a morphological microscope analysis is normally performed to select the best embryo to transfer, with the hope of obtaining a successful pregnancy. The morphological evaluation may combine number and size of blastomeres, fragmentation, multinucleation, blastocyst expansion, inner-cell mass and trophectoderm appearance. However, standard microscopy evaluation involves the removal of the embryos from the incubator, exposing them to changes in pH, temperature, and oxygen level. Additionally, morphological assessments might include high inter-observer variability. Recently, continuous embryo culture using time-lapse monitoring (TLM) has allowed embryologists to analyse the dynamic and morphokinetic events of embryo development and, based on that, the embryologist is able to scrutinize the complete sequence of embryonic evolution, from fertilization to the blastocyst formation. Therefore, TLM allows an uninterrupted culture condition, reducing the need to remove embryos from the incubator. The monitoring system is normally composed of a standard incubator with an integrated microscope coupled to a digital camera, which is able to collect images at regular times, and subsequently processed into video. These data can be annotated and analyzed using an integrated software, therefore this allows embryologists to facilitate the process of embryo selection for transfer. The main aim of this paper is to discuss the potential benefits and uses of the TLM in the embryology laboratory.

Culture conditions have a profound effect on the quality of in vitro-produced embryos. Co-culturing embryos with somatic cells has some beneficial effects on embryonic development. Considering the ability of stem cells to secrete a broad range of growth factors with different biological activities, we hypothesized that bovine amniotic membrane stem cells (bAMSCs) might be superior to bovine oviduct epithelial cells (BOECs) in supporting embryonic development and enhancing their cryo-survival. Bovine abattoir-derived oocytes were matured and fertilized in vitro. The resultant presumptive zygotes were then cultured up to the blastocyst stage in the following groups: (i) co-culture with bAMSCs, (ii) co-culture with BOECs, and (iii) cell-free culture (Con). Embryos that reached the blastocyst stage were vitrified and warmed, and their post-warming re-expansion, survival and hatching rates were evaluated after 72 h culture. Results showed that the cleavage, blastocyst, and 2 h post-warming re-expansion rates of embryos did not differ between groups. However, their survival rates in BOEC and bAMSC groups were significantly higher compared with the control (72.7, 75.6 and 37.5%, respectively, P < 0.05). In conclusion, our results showed that the cryo-survivability of IVF-derived bovine embryos could be improved through co-culturing with bAMSCs. Moreover, considering the possibility to provide multiple passages from bAMSCs compared with BOECs, due to their stemness properties and their ability to produce growth factors, the use of bAMSCs is a good alternative to BOECs in embryo co-culture systems.

Preeclampsia (PE), a pregnancy-specific disease, has become one of the leading causes of maternal and neonatal morbidity and mortality. Pathogenesis of PE has still not been fully addressed and there is a great need to develop early diagnosis markers and effective therapy. This study aimed to determine if lncRNA SNHG14 has a protective effect on placental trophoblast and prevents PE. SNHG14 levels in the peripheral blood from patients with PE or from women with healthy pregnancies were detected using RT-qPCR. The relationship between SNHG14 and miR-330-5p was determined using a dual-luciferase reporter assay. In addition, cell proliferation and cell cycle were evaluated by performing CCK8 assays and flow-cytometric analysis, respectively. Wound-healing and transwell assays were performed to assess cell migration and invasion ability. lncRNA SNHG14 was downregulated in PE patients; it was involved in trophoblast proliferation and regulated cell proliferation during G1/S transition. In addition, lncRNA SNHG14 promoted migration, invasion and epithelial–mesenchymal transition (EMT) in HTR-8/SVneo cells. Luciferase reporter assay indicated that lncRNA SNHG14 served as a molecular sponge for miR-330-5p and negatively regulated miR-330-5p expression in PE. Furthermore, the effects of silenced SNHG14 on trophoblast proliferation, migration, invasion and EMT were reversed by addition of miR-330-5p inhibitor, suggesting that in PE lncRNA SNHG14 functions by competitively binding to miR-330-5p. Taken together, the current study demonstrated that in PE lncRNA SNHG14 is a vital regulator by binding to miR-330-5p. SNHG14 might serve as a therapeutic application in PE progression.

Spermatogenesis is an androgen-dependent event, and testosterone is the major androgen source. The enzyme 5-alpha reductase converts testosterone to dihydrotestosterone (DHT) in testicular and peripheral tissues. Polymorphisms in genes encoding 5-alpha reductase may be associated with impaired male fertility. The present study aimed to investigate the relationship between 5-alpha reductase type 2 (SRD5A2) gene rs523349 polymorphism and non-obstructive azoospermia (NOA) in Turkish patients. The study included 75 NOA patients and 43 fertile men from Turkey. No significant relationship was found between SRD5A2 gene rs523349 polymorphism and male infertility (P = 0.071). There was a statistically significant difference in total testosterone level and total testis volume between NOA patients and the control groups, however there was no significant difference between serum follicle-stimulating hormone and luteinizing hormone levels. Our results showed that SRD5A2 gene rs523349 polymorphism was not associated with NOA in Turkish patients.

Chronic fatigue syndrome (CFS) is characterized by extreme fatigue and disabling symptoms. Women with CFS often have a high risk of gynaecological problems such as irregular menstruation, endometriosis and pelvic pain and sexual dysfunction. Our previous results have shown that, in pregnant mice, CFS significantly decreased the progestational hormone level in serum, as well as learning and memory, and the function of the hypothalamus–pituitary–gonadal axis. In addition, the F1 generation also suffered from congenital hypothyroidism. At present, there has been no report about placenta formation and embryonic development in pregnant mice with CFS. The aim of the present study was to investigate the influence of CFS on the morphology, oxidative stress and Wnt/β-catenin signalling pathway during placenta formation. In this study, we found that CFS decreased the number of implantation sites for blastocysts, and increased the number of absorbed, stillborn and malformed fetuses. The morphology and structure of the placenta were abnormal in pregnant mice with CFS. Further study found that the oxidative stress in serum, uterus and placenta was increased in pregnant mice with CFS, while the levels of antioxidase were decreased. CFS also inhibited the Wnt/β-catenin signalling pathway in the placenta. These results suggested that inhibition of the Wnt/β-catenin signalling pathway and enhanced oxidative stress play an important role in abnormal placentation in pregnant mice with CFS.

Ram spermatozoa are very sensitive to any cold shock or oxidative damage, therefore making them unsuitable for prolonged storage or distant transport to specialized laboratories for flow-cytometric analysis. The aim of this study was to stain ram semen samples with several fluorescent markers and analyse their stability during formaldehyde fixation. Briefly, freshly collected semen samples were stained for apoptosis (annexin V-FITC, YO-PRO™-1 and FLICA), acrosomal damage (PNA-AF488 and FITC-conjugated antibody against GAPDHS), mitochondrial activity (Mitotracker probes), oxidative damage [dihydroethidium (DHE) and CellROX™ Green] and cell viability (live/dead fixable viability dyes). Next, samples were fixed in buffer containing formaldehyde and then washed. Stained sample were analyzed using flow cytometer before fixation, immediately after fixation, and at 5 h and 20 h post-fixation. Fluorescent signals and the proportion of positively stained spermatozoa were compared statistically in fresh and post-fixed samples. All examined markers, except YO-PRO-1 (decreased significantly, P < 0.05), retained their fluorescence intensities after fixation. In conclusion, several tested markers were able to withstand formaldehyde fixation of ram semen samples as follows: annexin V and FLICA for apoptosis; PNA for acrosomal status; MitoTracker Red CMXRos for mitochondrial activity; and CellROX Green for oxidative status in combination with a suitable live/dead fixable viability dye. This optimized methodology could help to comprehensively analyse the quality of ram semen from local farms countrywide.

Human embryos generated in vitro have a high incidence of chromosomal abnormalities that negatively affect pregnancy rate. Embryos generated in vitro secrete extracellular vesicles (EVs) into the culture medium that could be used potentially as indicators of embryo competence. This research aimed to evaluate the concentration and size of EVs and their gDNA content as an indicator of developmental competence in human embryos. Human embryos generated by intracytoplasmic sperm injection (ICSI) were classified morphologically as of either TOP, FAIR or POOR quality. Culture medium and developmentally arrested embryos (which were not able to be used for embryo transfer) were collected. Microvesicles, exosomes (MV/Exo) and apoptotic bodies (ABs) were isolated from culture medium. Nanoparticle tracking analysis (NTA) and array comparative genomic hybridization (aCGH) analysis were performed to evaluate EVs and their gDNA content. From NTA, the diameter (mean) of MVs/Exo from TOP quality embryos was higher (112.17 nm) compared with that of FAIR (108.02) and POOR quality embryos (102.78 nm) (P < 0.05). aCGH analysis indicated that MVs/Exo and ABs carried gDNA with the presence of 23 chromosome pairs. However, when arrested embryos were compared with their respective MVs/Exo and ABs, the latter had an increased rate of chromosomal abnormalities (24.9%) compared with embryos (8.7%) (P < 0.05). In conclusion, the size of EVs from culture medium might be an alternative for evaluating competence of human embryos, however more studies are needed to validate the use of gDNA from EVs as an indicator of embryo competence.

The present study investigated the effects of c-type natriuretic peptide (CNP) on the development of murine preantral follicles during in vitro growth (IVG). Preantral follicles isolated from ovaries of Kunming mice were cultured in vitro. In the culture system, CNP was supplemented in the experimental groups and omitted in the control groups. In Experiment 1, CNP was only supplemented at the early stage and follicle development was evaluated. In Experiments 2 and 3, CNP was supplemented during the whole period of in vitro culture. In Experiment 2, follicle development and oocyte maturity were evaluated. In Experiment 3, follicle development and embryo cleavage after in vitro fertilization (IVF) were assessed. The results showed that in the control groups in all three experiments, granulosa cells migrated from within the follicle and the follicles could not reach the antral stage. In the experimental groups in all three experiments, no migration of granulosa cells was observed and follicle development was assessed as attaining the antral stage, which was significantly superior to that of the control group (P < 0.0001). Oocyte meiotic arrest was effectively maintained, hence giving good developmental competence. In conclusion, CNP supplementation in the culture system during IVG benefited the development of murine preantral follicles.

This research aimed to examine the effects of thyme, celery and salinomycin on ovarian sex hormones, reproductive traits and antioxidant status during the estrous cycle. Seventy-five mature Barki ewes aged 2–3 years with an average weight of 40 ± 1.5 kg were assigned randomly into five groups (15 head/group). Group 1 was kept as the control; groups 2 and 3 received 20 g/head/day thyme (T) and celery (C) as dried herbs, respectively. Group 4 (T×C) received 10 g thyme + 10 g celery/head/day, and group 5 was treated with salinomycin 1 g/head/day. Blood samples were collected during follicular and luteal phases of the estrous cycle. Thyme and celery and the mixture of T×C increased (P < 0.01) estradiol-17β (E2) during the follicular phase of the estrous cycle, while only the celery group showed a marked (P < 0.001) increase in progesterone (P4) during the luteal phase compared with the control. Salinomycin supplementation decreased (P < 0.05) E2 concentrations during the follicular and luteal phases of the estrous cycle. Supplementation with thyme and celery enhanced (P < 0.001) antioxidant capacity in the luteal phase compared with the follicular stage. The salinomycin group showed increased (P < 0.01) levels of reduced glutathione (GSH) and decreased malondialdehyde (MDA) levels compared with the control group throughout luteal phase. For the interaction between estrous phases and treatments, thyme, celery, and T×C supplementation revealed an increase (P < 0.05) in superoxide dismutase (SOD), GSH, and glutathione disulfide (GSSG) levels compared with the control group during the follicular and luteal phases. Thyme and celery supplementation improved the number of services per conception and fertilization from 1st and 2nd inseminations, respectively. In conclusion, the applied treatment had significant effects on reproductive performance and antioxidant status in ewes throughout the estrous cycle.

The aim of this study was to investigate the effect of cyanocobalamin supplementation on in vitro maturation (IVM), in vitro fertilization (IVF), and subsequent embryonic development competence to the blastocyst stage, and in vitro development of mouse 2-cell embryos. Cumulus cells were prepared from mouse cumulus–oocyte complexes (COCs) and incubated for 24 h in an in vitro culture (IVC) medium that contained different concentrations of cyanocobalamin (100, 200, 300 or 500 pM). We collected 2-cell embryos from superovulated NMRI mice and cultured them in the same concentrations of cyanocobalamin (100, 200, 300 or 500 pM). After 42 h of IVM, we observed significantly increased oocyte maturation in the 200 pM cyanocobalamin-treated group compared with the control group (P < 0.0001). Mature oocytes cultured in 200 pM cyanocobalamin were fertilized and cultured in IVC medium with cyanocobalamin (100, 200, 300 or 500 pM) during early embryogenesis. The matured oocytes that were cultured in 200 pM cyanocobalamin had significantly higher 2-cell development rates compared with the control oocytes (P < 0.01). Embryos obtained from in vitro mature oocytes and in vivo fertilized oocytes that were cultured in 200 pM cyanocobalamin had significantly greater frequencies of development to the blastocyst stage and a significant reduction in 2-cell blocked and degenerated embryos compared with the control embryos (P < 0.0001). Embryos derived from oocytes fertilized in vivo with 200 pM cyanocobalamin had a higher percentage of blastocyst embryos compared with those derived from matured oocytes cultured in vitro (P < 0.0001). These finding demonstrated that the effects of cyanocobalamin on oocyte maturation, fertilization, and embryo development in mice depend on the concentration used in IVC medium.

Meiosis is a highly conserved process, and is responsible for the production of haploid gametes and generation of genetic diversity. We previously identified the transferrin receptor (TFRC) in the proteome profile of mice neonatal testes, indicating the involvement of the TFRC in meiosis. However, the exact molecular role of the TFRC in meiosis remains unclear. In this study, we aimed to determine the function of the TFRC in neonatal testicular development by TFRC knockdown using the testis culture platform. Our results showed high TFRC expression in 2-week testes, corresponding to the first meiotic division. Targeting TFRC using morpholino oligonucleotides resulted in clear spermatocyte apoptosis. In addition, we used the chromosomal spread technique to show that a deficiency of TFRC caused the accumulation of leptotene and zygotene spermatocytes, and a decrease of pachytene spermatocytes, indicating early meiotic arrest. Moreover, the chromosomes of TFRC-deficient pachytene spermatocytes displayed sustained γH2AX association, as well as SYCP1/SYCP3 dissociation beyond the sex body. Therefore, our results demonstrated that the TFRC is essential for the progression of spermatocyte meiosis, particularly for DNA double-stranded break repair and chromosomal synapsis.

Alcohol consumption has long been shown to affect both fetal health and pregnancy. In this study, antral follicle count, maturation level of oocytes including morphological assessment and number of metaphase I (MI), metaphase II (MII) and germinal vesicle (GV) stage oocytes obtained from young women (age < 30 years old) with or without alcohol consumption were investigated. In total, 20 healthy women who were social drinkers and 36 healthy women who do not consume alcohol were involved in this study. Women in both study and control groups were undergoing controlled ovarian stimulation. The antral follicle count and the number and quality of the oocytes retrieved were evaluated and recorded. In total, 635 antral follicles, 1098 follicles and 1014 oocytes with 820 MII, 72 MI and 78 GV stage oocytes were collected from the social drinkers. In the control group, 628 antral follicles, 1136 follicles and 1085 oocytes with 838 MII, 93 MI and 102 GV stage oocytes were evaluated. The results of this study showed that the antral follicle count was very similar in both groups. The number of oocytes and MII stage oocytes was slightly higher in the control group, although it was not a significant difference. This study showed that although the consumption of alcohol may have adverse effects post-implantation, it may not have a solid effect during oogenesis in young women. The results of this study are especially important in clinical settings as some women who are social drinkers undergo in vitro fertilization treatments.