Microbial contamination in assisted reproductive technology: source, prevalence, and cost.

Even the strictest laboratories and clinics are prone to the occurrence of microbial contamination. In the case of in vitro fertilization (IVF) research and practice facilities, the number of possible sources is particularly vast. In addition to ambient air, personnel, and non-sterilized materials, follicular fluid and semen from patients are a very common gateway for a diverse range of bacteria and fungi into embryo cultures. Even so, reports of contamination cases are rare, what leads many clinics to see the issue as a negligible risk. Microbiological contamination may result in the demise of the patient's embryos, leading to additional costs to both the patient and the clinics. Regardless of financial loss, emotional costs, and stress levels during IVF are highly distressing. Other worrisome consequences include DNA fragmentation, poor-quality embryos, early pregnancy loss or preterm birth, and possible long-term damages that need further investigation. In this review, we aimed to shed a light on the issue that we consider largely underestimated and to be the underlying cause of poor IVF outcomes in many cases. We also discuss the composition of the microbiome and how its interaction with the reproductive tract of IVF-seeking patients might influence their outcomes. In conclusion, we urge clinics to more rigorously identify, register, and report contamination occurrences, and highlight the role of the study of the microbiome to improve overall results and safety of assisted reproduction.

An update on the aspects of Zika virus infection on male reproductive system.

Zika virus (ZIKV) is mainly transmitted through Aedes mosquito bites, but sexual and post-transfusion transmissions have been reported. During acute infection, ZIKV is detectable in most organs and body fluids including human semen. Although it is not currently epidemic, there is a concern that the virus can still reemerge since the male genital tract might harbor persistent reservoirs that could facilitate viral transmission over extended periods, raising concerns among public health and assisted reproductive technologies (ART) experts and professionals. So far, the consensus is that ZIKV infection in the testes or epididymis might affect sperm development and, consequently, male fertility. Still, diagnostic tests have not yet been adapted to resource-restricted countries. This manuscript provides an updated overview of the cellular and molecular mechanisms of ZIKV infection and reviews data on ZIKV persistence in semen and associated risks to the male reproductive system described in human and animal models studies. We provide an updated summary of the impact of the recent ZIKV outbreak on human-ART, weighing on current recommendations and diagnostic approaches, both available and prospective, with special emphasis on mass spectrometry-based biomarker discovery. In the light of the identified gaps in our accumulated knowledge on the subject, we highlight the importance for couples seeking ART to follow the constantly revised guidelines and the need of specific ZIKV diagnosis tools for semen screening to contain ZIKV virus spread and make ART safer.

Effects of angiotensin II, atrial natriuretic peptide and endothelin-1 on proliferation and steroidogenic output of bovine granulosa cells cultured in a chemically defined system.

The role of local factors in the modulation of granulosa cell (GC) proliferation and differentiation is well described in the literature. The present work used a long-term bovine GC culture, in chemically defined medium without gonadotropins, to study the effects of angiotensin II (Ang II), atrial natriuretic peptide (ANP) and endothelin-1 (EDN1) on the steroidogenesis and cellular proliferation. Small follicles (3-5mm in diameter) from ovaries obtained in the slaughterhouse were selected according to their vascularization and follicular fluid color in order to isolate GC. Granulosa cells were plated at a density of 5×10(4)cells/well in supplemented alpha-MEM containing 3 levels (0, 10(-8)M and 10(-7)M) of Ang II, ANP, and EDN1 for up to 96h. Proliferation was evaluated by tritiated thymidine incorporation. The results showed that Ang II, ANP, and EDN1 modulate the steroidogenic output and proliferation index of GCs depending on the dose and time of culture. The selected vasoactive peptides increased androstenedione (A4) consumption in parallel with increased estradiol (E2). Although the peptides also promoted a significant increase in pregnenolone (P5) and progesterone (P4) production, the E2:P4 ratio was maintained at a high at most of the tested doses. Taken together, our in vitro data suggest that these vasoactive factors may have a direct effect on physiological follicular deviation, favoring dominance of the selected follicle.

Exposing bovine cumulus-oocyte complexes to aromatizable androgen restore serum-induced low estradiol production in vitro.

We aimed in this study to assess whether serum-decreased bovine cumulus-oocyte complex (COC) steroidogenesis during in vitro maturation (IVM) is caused by deficient androgen milieu. For this approach, bovine COCs were cultured in serum-supplemented IVM medium in the presence or absence of 1 µM androstenedione. After 24 h of culture, medium was collected and analyzed for its content of estradiol-17ß (E2) and progesterone (P4). Medium E2 content markedly increased after incubation of COCs with androstenedione (17.52 ± 1.86 ng/ml to the androgen group; 0.32 ± 0.05 ng/ml to the non-androgen group). No significant difference in the P4 content was detected despite the presence of androstenedione (21.83 ± 1.61 ng/ml to the androgen group; 21.73 ± 1.67 ng/ml to the non-androgen group). Our data provide compelling evidence that bovine COCs steroidogenesis remains functional during culture in serum-supplemented medium and suggest that serum-induced decreased COCs estradiol secretion is caused by deficiency of an aromatizable androgen source.

Preventing Microbial Contamination during Long-Term In Vitro Culture of Human Granulosa-Lutein Cells: An Ultrastructural Analysis.

Purpose. To investigate whether the addition of antibiotic/antimycotic during human granulosa-lutein cells (GLCs) isolation and cell-plating procedures prevents microbial contamination after 144 h of culture and also evaluate the effects of contamination on GLCs ultrastructure and steroid secretion. Methods. GLCs obtained from five women submitted to assisted reproductive techniques (ARTs) were isolated with PBS supplemented with antibiotic/antimycotic or PBS nonsupplemented and cultured for 144 h. GLCs were evaluated by transmission electron microscopy (TEM), and estradiol (E2) and progesterone (P4) secretion was assayed by chemiluminescence. Results. Although no contaminating microorganisms were identified by light microscopy, TEM analyses revealed several bacterial colonies in culture dishes of GLCs isolated with only PBS. Bacterial contamination disrupted the adherence of the GLCs to the culture plate interfering with monolayer formation affecting the growth pattern of GLCs. Various cellular debris and bacteria were observed, and no organelles were found in the cytoplasm of infected cells. While bacterial contamination decreased estradiol media levels, it increased progesterone, as compared with noncontaminated group. Conclusion. Taken together, our data showed that the addition of a high dose of antibiotic/antimycotic during the isolation and cell-plating procedures prevents microbial contamination of long-term GLCs culture as its effects on cells growth and function in vitro.

The low fertility of repeat-breeder cows during summer heat stress is related to a low oocyte competence to develop into blastocysts.

It was hypothesized the lower fertility of repeat-breeder (RB) Holstein cows is associated with oocyte quality and this negative effect is enhanced during summer heat stress (HS). During the summer and the winter, heifers (H; n=36 and 34, respectively), peak-lactation (PL; n=37 and 32, respectively), and RB (n=36 and 31, respectively) Holstein cows were subjected to ovum retrieval to assess oocyte recovery, in vitro embryonic developmental rates, and blastocyst quality [terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and total cell number]. The environmental temperature and humidity, respiration rate, and cutaneous and rectal temperatures were recorded in both seasons. The summer HS increased the respiration rate and the rectal temperature of PL and RB cows, and increased the cutaneous temperature and lowered the in vitro embryo production of Holstein cows and heifers. Although cleavage rate was similar among groups [H=51.7% ± 4.5 (n=375), PL=37.9% ± 5.1 (n=390), RB=41.9% ± 4.5 (n=666)], blastocyst rate was compromised by HS, especially in RB cows [H=30.3% ± 4.8 (n=244) vs. 23.3% ± 6.4 (n=150), PL=22.0% ± 4.7 (n=191) vs. 14.6% ± 7.6 (n=103), RB=22.5% ± 5.4 (n=413) vs. 7.9% ± 4.3 (n=177)]. Moreover, the fragmentation rate of RB blastocysts was enhanced during the summer, compared with winter [4.9% ± 0.7 (n=14) vs. 2.2% ± 0.2 (n=78)] and other groups [H=2.5% ± 0.7 (n=13), and PL=2.7% ± 0.6 (n=14)] suggesting that the association of RB fertility problems and summer HS may potentially impair oocyte quality. Our findings provide evidence of a greater sensitivity of RB oocytes to summer HS.

Cytoplasmic maturation of bovine oocytes: structural and biochemical modifications and acquisition of developmental competence.

Oocyte maturation is a long process during which oocytes acquire their intrinsic ability to support the subsequent stages of development in a stepwise manner, ultimately reaching activation of the embryonic genome. This process involves complex and distinct, although linked, events of nuclear and cytoplasmic maturation. Nuclear maturation mainly involves chromosomal segregation, whereas cytoplasmic maturation involves organelle reorganization and storage of mRNAs, proteins and transcription factors that act in the overall maturation process, fertilization and early embryogenesis. Thus, for didactic purposes, we subdivided cytoplasmic maturation into: (1) organelle redistribution, (2) cytoskeleton dynamics, and (3) molecular maturation. Ultrastructural analysis has shown that mitochondria, ribosomes, endoplasmic reticulum, cortical granules and the Golgi complex assume different positions during the transition from the germinal vesicle stage to metaphase II. The cytoskeletal microfilaments and microtubules present in the cytoplasm promote these movements and act on chromosome segregation. Molecular maturation consists of transcription, storage and processing of maternal mRNA, which is stored in a stable, inactive form until translational recruitment. Polyadenylation is the main mechanism that initiates protein translation and consists of the addition of adenosine residues to the 3' terminal portion of mRNA. Cell cycle regulators, proteins, cytoplasmic maturation markers and components of the enzymatic antioxidant system are mainly transcribed during this stage. Thus, the objective of this review is to focus on the cytoplasmic maturation process by analyzing the modifications in this compartment during the acquisition of meiotic competence for development.

Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocysts derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules.

In vitro culture conditions affect both the maternal and embryonic expression of genes and is likely to alter both oocyte and embryo developmental competence. The search for better and less variable culture conditions simulating those in vivo has led to the development of defined culture media, with lower impact on the molecular reprogramming of oocytes and embryos. We evaluated embryo development and relative abundance (RA) of Hsp-70 and Bax transcripts in bovine blastocysts produced from oocytes matured in a chemically defined IVM system with synthetic polymers. Immature cumulus oocyte complexes (COCs) were matured for 22-24h in alpha-MEM supplemented with IGF-1, insulin, 0.1% polyvinyl alcohol (PVA), or 0.1% polyvinylpyrrolidone (PVP), but without FSH or LH. The control group consisted of COCs matured in TCM plus FSH and 10% estrous cow serum. After fertilization, presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to transcript analysis by real-time PCR. Cleavage rate was higher (P<0.05) for the control group (68.3%) than for the PVA (54.4%) and PVP-40 (58.3%) groups. Nevertheless, there was no difference among the PVA, PVP-40 and control groups in blastocyst or hatching rates. Similarly, no difference in relative abundance of Hsp-70 and Bax transcripts was detected in comparison to the control group. We inferred that bovine oocytes can be matured in serum- and gonadotrophin-free medium supplemented with PVA or PVP, enriched with IGF-I and insulin, without altering post-cleavage development and relative abundance of some genes associated with stress and apoptosis.

Evaluation of the toxicity of Solanum lycocarpum in the reproductive system of male mice and rats.

Powdered Solanum lycocarpum fruit is commonly used to treat diabetes, but apparently no studies have been conducted to evaluate potential adverse side effects. In the present paper the toxic effect of S. lycocarpum was evaluated in adult male Wistar rats and Swiss mice. The administration of an aqueous extract prepared using a powder obtained from the S. lycocarpum fruit at two different dose levels (60 mg/15 ml and 120 mg/15 ml distilled water for rats and 30 mg/15 ml and 60 mg/15 ml distilled water for mice, twice daily for 5 days in each case) did not produce body weight variations in either species although a significant weight change was observed in some organs. Significant weight loss was observed only in the ventral prostate of mice receiving the high dose treatment. These results suggest a toxic effect of S. lycocarpum on the male reproductive system of the Swiss mouse, with possible antiandrogenic activity, but there was no apparent antifertility activity in rats at the doses given.