The presence of Leptospira spp. in the follicular fluid of naturally infected cows affects the overall efficiency of the in vitro embryo production technique.

The relationship between Leptospira infection and reproductive failures, as well as the mechanisms that lead to it, has not yet been fully established. It has been hypothesized that the presence of Leptospira spp. in the follicular fluid (FF) could impair the oocyte developmental competence. Thus, the impact of the presence of Leptospira spp. in the FF on in vitro embryo production (IVEP) outcomes was assessed. Dairy cows (n=244) from different farms were subjected to ovum pick-up for cumulus-oocyte complexes (COCs) collection. After PCR analysis of the FF, cows were retrospectively allocated into either: positive (POS-FF) or negative (NEG-FF) group. Statistical modeling was conducted using the farm, PCR result, and laboratory in which the IVEP was performed as effects. Noteworthy, 26.6% of the animals were positive for Leptospira spp., and 70% of farms had at least one POS-FF cow in the herd. POS-FF cows had a lower number of COCs recovered (22.6 ± 1.2 vs 15.0 ± 2.8, P=0.036), rate of viable COCs (85.6 ± 0.9% vs 78.1 ± 2.8%, P=0.015), number of good-quality COCs (16.0 ± 0.9 vs 9.8 ± 2.1, P=0.026), cleaved embryos (11.9 ± 0.7 vs 7.5 ± 1.5, P=0.032), and blastocysts (7.3 ± 0.4 vs 2.3 ± 0.7, P=0.044) yielded per cow. In conclusion, the presence of Leptospira spp. in the FF of naturally infected cows impaired the amount of COCs recovered, decreasing the overall IVEP efficiency.

Microbiology of Human Follicular Fluid and the Vagina and Its Impact on in Vitro Fertilization Outcomes.

PURPOSE: The present study aimed to identify microorganisms in follicular fluids and to investigate their association with in vitro fertilization (IVF) outcomes. MATERIALS AND METHODS: This study was conducted as a prospective study of 49 infertile females undergoing IVF/intracytoplasmic sperm injection cycles between 2013 and 2016. Paired follicular fluid and vaginal secretions were collected on the day of ovum pick up and were cultured to detect microorganisms. RESULTS: Fifteen women (30.6%) had no microorganisms in follicular fluid or vaginal swabs, 23 (46.9%) had microorganisms on vaginal swab alone, 3 (6.1%) had microorganisms in follicular fluid alone, and 8 (16.3%) had microorganisms in both follicular fluid and vaginal swabs. The same microorganisms were detected in both the follicular fluid and vaginal swabs of three women, while different microorganisms were detected between follicular fluid and vaginal swabs in five women. Follicular fluid microorganisms were not associated with embryo quality or clinical pregnancy rates during IVF cycles. However, significantly decreased implantation rates (9.1% vs. 29.4%, p=0.031) and clinical pregnancy rates on embryo transfer day 5 (0% vs. 83.3%, p=0.048) were observed in the group that was positive for vaginal pathogens. CONCLUSION: Follicular fluid contains microorganisms that can differ from those in the vagina of the same women; however, they do not appear to be associated with embryo quality or clinical pregnancy rates in IVF cycles. In contrast, vaginal pathogens were found to be associated with worse implantation rates and clinical pregnancy rates in IVF cycles.

Characterization of leptospiral DNA in the follicular fluid of non-pregnant cows.

BACKGROUND: Bovine leptospirosis is mainly characterized by reproductive disorders. Contamination of the oocyte was previously demonstrated in vitro, resulting in some apparent damage. However, it is not clear whether it occurs under natural conditions. The present study aimed to characterize the presence of pathogenic Leptospira DNA in the ovarian follicles of non-pregnant cows. METHODS: Follicular fluid samples were collected from 65 animals and subjected to lipL32 PCR and secY sequencing. RESULTS: In total, seven of 65 (10.8%) were positive, indicating a possible early infection of the oocyte. Moreover, secY sequencing identified L. interrogans and L. santarosai, both very closely related to bovine strains from the Sejroe serogroup (Hardjoprjitno and Guaricura). We demonstrated that ovarian follicles can also be infected. CONCLUSIONS: It was hypothesised that ovarian infection can contribute to embryonic death, causing reproductive failure and estrus repetition. In the present study, we show that the organism identified in the follicle is closely related to one that is known to be associated with reproductive disorders.

The presence of microorganisms in follicular fluid and its effect on the outcome of in vitro fertilization-embryo transfer (IVF-ET) treatment cycles.

OBJECTIVES: To determine if the presence of microorganisms in follicular fluid of women undergoing in vitro fertilization-embryo transfer (IVF-ET) adversely affects the outcome of the treatment cycles. METHODS: Follicular fluid was collected from 86 women enrolled for IVF-ET at the National Hospital, Abuja from June 1, 2018 to December 1, 2018. Microscopy imaging and cultures were performed to identify bacteria and fungi in the follicular fluid and the vagina. Women with follicular fluid microorganisms were the test group while the controls were those without follicular fluid microorganisms. Fertilization and pregnancy rates were subsequently determined and their association with the presence of follicular fluid microorganisms was assessed using univariable and multivariable logistic regression modelling. RESULTS: The mean age and mean Body Mass Index (BMI) of the participants were 35(± 3.5) years and 28(± 4.9) kg/m2 respectively. Bacteria and fungi were isolated in 17% of the follicular fluid samples collected (n = 15/86). Most common isolates were Streptococcus spp. (n = 4/15), Staphylococcus aureus (n = 7/15), Enterococcus spp. (n = 4/15), Lactobacillus species (n = 2/15) and Candida albicans (n = 2/15). There was no statistically significant difference in the fertilization rates (adjusted odds ratio [AdjOR] 0.55, 95% confidence interval [CI] 0.04-7.34; P = 0.10) and pregnancy rates (AdjOR 4.02, 95% CI 0.56-28.92; P = 0.19) between the group of women with positive follicular fluid bacterial /fungal colonization as compared against those with negative follicular fluid colonization. CONCLUSION: Isolation of microorganisms from follicular fluid did not adversely affect fertilization and pregnancy rates following IVF-ET treatment cycles at the fertility centre of National Hospital, Abuja.

Detection of bacteria in bovine ovarian follicular fluid.

The aim of this study was to examine the sterility of follicular fluid in large ovarian follicles in dairy cows. In all, 17 samples of paired follicular fluids and uterine contents collected from post-slaughtered dairy cows were cultured to detect aerobic and facultative anaerobic bacteria. Furthermore, the origin of the bacterial isolates from samples of follicular fluid and the uteri was also investigated using PFGE analysis. Follicular fluid concentrations of lipopolysaccharides were also determined. Of 17 uterine samples, 15 (88%) were detected as contaminated. In total, nine different bacterial genera and species were identified in the uterine and follicular fluid samples. Escherichia coli was the most prevalent bacterial species isolated from the uterine samples. Out of seven isolates of Staphylococcus aureus from the uterine samples, 6 (85%) were coagulase positive. Six isolates of Staphylococcus spp. were identified in 6 out of 17 follicular fluid samples (35%). Two out of six isolates were identified as Staphylococcus aureus (33%). Our results show that ovarian follicular fluid is not sterile in the bovine. The presence of Staphylococcus aureus in follicular fluid may partly explain the occurrence of infertility in some dairy cows. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the present study show that ovarian follicular fluid is not sterile in bovines. The presence of Staphylococcus aureus in follicular fluid may partly explain the occurrence of infertility in some dairy cows.

TUNEL analysis of DNA fragmentation in mouse unfertilized oocytes: the effect of microorganisms within human follicular fluid collected during IVF cycles.

Recently we reported the presence of bacteria within follicular fluid. Previous studies have reported that DNA fragmentation in human spermatozoa after in vivo or in vitro incubation with bacteria results in early embryo demise and a reduced rate of ongoing pregnancy, but the effect of bacteria on oocytes is unknown. This study examined the DNA within mouse oocytes after 12 hours' incubation within human follicular fluids (n=5), which were collected from women undergoing in vitro fertilization (IVF) treatment. Each follicular fluid sample was cultured to detect the presence of bacteria. Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) was used to label DNA fragmentation in ovulated, non-fertilized mouse oocytes following in vitro incubation in human follicular fluid. The bacteria Streptococcus anginosus and Peptoniphilus spp., Lactobacillus gasseri (low-dose), L. gasseri (high-dose), Enterococcus faecalis, or Propionibacterium acnes were detected within the follicular fluids. The most severe DNA fragmentation was observed in oocytes incubated in the follicular fluids containing P. acnes or L. gasseri (high-dose). No DNA fragmentation was observed in the mouse oocytes incubated in the follicular fluid containing low-dose L. gasseri or E. faecalis. Low human oocyte fertilization rates (<29%) were associated with extensive fragmentation in mouse oocytes (80-100%). Bacteria colonizing human follicular fluid in vivo may cause DNA fragmentation in mouse oocytes following 12h of in vitro incubation. Follicular fluid bacteria may result in poor quality oocytes and/or embryos, leading to poor IVF outcomes.

Microorganisms within human follicular fluid: effects on IVF.

Our previous study reported microorganisms in human follicular fluid. The objective of this study was to test human follicular fluid for the presence of microorganisms and to correlate these findings with the in vitro fertilization (IVF) outcomes. In this study, 263 paired follicular fluids and vaginal swabs were collected from women undergoing IVF cycles, with various causes for infertility, and were cultured to detect microorganisms. The cause of infertility and the IVF outcomes for each woman were correlated with the microorganisms detected within follicular fluid collected at the time of trans-vaginal oocyte retrieval. Microorganisms isolated from follicular fluids were classified as: (1) 'colonizers' if microorganisms were detected within the follicular fluid, but not within the vaginal swab (at the time of oocyte retrieval); or (2) 'contaminants' if microorganisms detected in the vagina at the time of oocyte retrieval were also detected within the follicular fluid. The presence of Lactobacillus spp. in ovarian follicular fluids was associated with embryo maturation and transfer. This study revealed microorganisms in follicular fluid itself and that the presence of particular microorganisms has an adverse affect on IVF outcomes as seen by an overall decrease in embryo transfer rates and pregnancy rates in both fertile and infertile women, and live birth rates in women with idiopathic infertility. Follicular fluid microorganisms are a potential cause of adverse pregnancy outcomes in IVF in both infertile women and in fertile women with infertile male partners.

Hormone-dependent bacterial growth, persistence and biofilm formation--a pilot study investigating human follicular fluid collected during IVF cycles.

Human follicular fluid, considered sterile, is aspirated as part of an in vitro fertilization (IVF) cycle. However, it is easily contaminated by the trans-vaginal collection route and little information exists in its potential to support the growth of microorganisms. The objectives of this study were to determine whether human follicular fluid can support bacterial growth over time, whether the steroid hormones estradiol and progesterone (present at high levels within follicular fluid) contribute to the in vitro growth of bacterial species, and whether species isolated from follicular fluid form biofilms. We found that bacteria in follicular fluid could persist for at least 28 weeks in vitro and that the steroid hormones stimulated the growth of some bacterial species, specifically Lactobacillus spp., Bifidobacterium spp. Streptococcus spp. and E. coli. Several species, Lactobacillus spp., Propionibacterium spp., and Streptococcus spp., formed biofilms when incubated in native follicular fluids in vitro (18/24, 75%). We conclude that bacteria aspirated along with follicular fluid during IVF cycles demonstrate a persistent pattern of growth. This discovery is important since it can offer a new avenue for investigation in infertile couples.

Microbial colonization of follicular fluid: alterations in cytokine expression and adverse assisted reproduction technology outcomes.

BACKGROUND: Previous studies have measured cytokines expressed within follicular fluid and compared the profiles with the aetiology of infertility and/or successful or unsuccessful assisted reproduction technology (ART) outcomes. METHODS: In this study, 71 paired follicular fluid and vaginal secretions collected from ART patients were cultured to detect microorganisms and tested for the presence of cytokines. Patient specimens were selected for assay based on two criteria: whether the follicular fluid specimen was colonized (with microorganisms prior to oocyte retrieval) or contaminated by vaginal flora and; the aetiology of infertility. Patients included fertile women (with infertile male partners; n = 18), women with endometriosis (n = 16) or polycystic ovary syndrome (PCOS, n = 14), or couples with a history of genital tract infection (n = 9) or idiopathic infertility (n = 14). RESULTS: Microorganisms and cytokines were detected within all tested specimens. Colonizing microorganisms in follicular fluid were associated with: decreased fertilization rates for fertile women (P = 0.005), women with endometriosis (P = 0.0002) or PCOS (P = 0.002) compared with women whose follicular fluid was contaminated at the time of oocyte retrieval and with decreased pregnancy rates for couples with idiopathic infertility (P = 0.001). A single cytokine was discriminatory for women with an idiopathic aetiology of infertility (follicular fluid interleukin (IL)-18). Unique cytokine profiles were also associated with successful fertilization (IL-1α, IL-1ß, IL-18 and vascular endothelial growth factor). CONCLUSIONS: Follicular fluid is not sterile. Microorganisms colonizing follicular fluid and the ensuing cytokine response could be a further as yet unrecognized cause and/or predictor of adverse ART outcomes and infertility.

Ovarian hydrobursitis in female camels (Camelus dromedaries): biochemical, bacterial and protozoal evaluation.

The aim of this study was to evaluate female camels affected with ovarian hydrobursitis (n = 31) for hematological and biochemical findings and for bacterial and protozoal infections. Blood samples were obtained and surgical ablation of the affected bursa was performed. Bursal fluid, follicular fluid, and serum were subjected to hormonal and biochemical analyses. Bursal fluids were cultured and colonies were identified using BioMérieux Vitek two compact system. Passive haemagglutination test was used for detection of Trypanosoma evansi. Indirect ELISA technique was carried out for detection of anti-Hydatid cysts anti-bodies. Neutrophilia was found in the affected animals (P = 0.01) with tendencies for monocytosis (P = 0.06) and eosinophelia (P = 0.05). Bursal fluid had a tendency for high estradiol-17ß concentration compared to blood serum (P = 0.07). Progesterone and cholesterol concentrations were similar in bursal fluid, follicular fluid and serum. Total protein, phosphorus, and magnesium concentrations were greater (P < 0.05) in the bursal fluid than in serum. Oligella urethralis, Alloiococcus otitis, Granulicatella adicens, Escherichia coli, Sphingobacterium thalpophilum, Streptococcus sanguinis, Aeromonas salmonicida, Pseudomonas stutzeri, Staphylococcus warneri, Staphylococcus hominis, and Rhizobium radiobacter were isolated from 46.7% of bursal fluids. T. evansi was positive in 9.7% of cases. None were positive for hydatid cyst. Accordingly, we suggest that the ovarian hydrobursitis syndrome is initially an inflammatory process and the accumulated bursal fluid is partially originated from follicular fluid.

Autoimmune response to Chlamydia trachomatis infection and in vitro fertilization outcome.

This observational study was conducted in 235 patients undergoing IVF who had a cervical swab positive for Chlamydia trachomatis and who underwent antibiotic treatment until a negative cervical swab before IVF attempt. After oocyte retrieval, follicular fluids of 109 patients out of 228 still showed the presence of IgA antichlamydia antibodies and a significantly lower pregnancy and implantation rate; therefore we conclude that patients should undergo IVF procedure after serum antichlamydia IgA tests negative.

[Detection of chlamydia antibodies in non-standard biological fluids in women with fertility disorders]. / Urcení antichlamydiových protilátek v nestandardních biologických tekutinách u zen s poruchami plodnosti.

OBJECTIVE: Detection of IgA and IgG antibodies against Chlamydia trachomatis in various biological materials in patients with fertility disorders. DESIGN: Retrospective study. SETTING: Department of Gynecology and Obstetrics, Medical Faculty of Charles University and Faculty Hospital, Plzen. METHODS: Antichlamydial antibodies IgA and IgG were detected in 73 patients. Examination of sera was performed in all 73 women, peritoneal fluid was examined in 20 patients who underwent diagnostic laparoscopy, follicular fluid samples in 22 women from IVF programme and antibodies in cervical ovulatory mucus were examined in 31 patients of the Division of Immunology of Reproduction. Results were compared with clinical findings and data of personal histories. Blood donors sera examination were used as a control group. RESULTS: High number of chlamydial antibody positivity was found in follicular fluid--in IgA (18/22, 82%) and IgG (16/22, 73%) and also in ovulatory cervical mucus in IgA (13/31, 42%). Examinations of sera did not differ significantly from the control group. CONCLUSION: High levels of chlamydial antibodies in follicular fluid samples could display connection between chlamydia infection and infertility, mainly as its organic cause (adhesions, tubal factor). IgA positivity in cervical ovulatory mucus is caused by local antibody production. Serologic examination of chlamydial antibodies is one of the most suitable examinations of entry in infertile patients as well as in pregnant women.

Detecçäo de células escamosas vaginais no aspirado de fluido folicular durante fertilizaçäo in vitro / Vaginal squamous cells detected in the follicular fluid aspirated during in vitro fertilization

RETROSPECTIVA: A possibilidade de contaminaçao dos folículos ovarianos puncionados para FIV pela flora bacteriana vaginal pode ser uma desvantagem dessa técnica. OBJETIVOS: Confirmar a presença de células escamosas vaginais no fluido folicular e estudar os possíveis efeitos nos oócitos recrutados através de sua capacidade de fertilizaçao. CASUISTICA E METODOLOGIA: Participaram desse estudo 8 pacientes incluídas no programa de FIV. Os fluidos obtidos por punçao transvaginal dos 3 primeiros folículos de cada ovário foram imediatamente enviados para análise citológica e bacteriológica. Os oócitos encontrados nesses fluidos foram analisados e classificados. Posteriormente foi observada a presença ou nao de fertilizaçao após a uniao com os espermatozóides dos respectivos maridos. RESULTADOS: As células vaginais estiveram presentes em 93,7 por cento dos primeiros folículos, 56,2 por cento dos segundos folículos e em 20 por cento dos terceiros folículos aspirados. A presença de células escamosas vaginais nos fluidos foliculares nao interferiu na capacidade de fertilizaçao dos oócitos. CONCLUSOES: A confirmaçao da presença de células vaginais no fluido folicular durante a punçao transvaginal para recrutamento de oócitos, em maior porcentagem nos folículos inicialmente puncionados, sugere a possibilidade de contaminaçao pélvica pelo meio vaginal. Entretanto, nao houve interferência sobre a taxa de fertilizaçao.

Evidencia de actividad antimicrobiana del líquido folicular: estudio evaluatorio y analítico de muestras obtenidas en un programa de reproducción asistida / Evidence of antimicrobial activity of follicular fluid: evaluatory and analytical study of samples obtained in an assisted reproduction program

Ante la evidente ausencia de infección en pacientes a quienes se les realiza punción ovárica para captura ovular se evaluó la hipótesis de la posible acción antibacteriana que pudiera existir en el líquido folicular. Por lo anterior se tomaron 110 muestras de dicho fluido para análisis antibacteriano de las cuales fueron estudiadas 37, por ser las más claras y no contaminadas. Dichas muestras fueron obtenidas de pacientes hiperestimuladas que estaban a captura ovular a través de vagina por medio de guía ultrasonográfica. Se preparó inóculo bacteriana para evaluar la actividad antimicrobiana del líquido folicular contra cepas de E. coli, P. aeuroginosa, S. agalactie, L. monicitogenesis y C. albicans. Se efectuó recuento de colonias bacterianas a la 2,4,6,12 y 24 horas. El resumen de los resultados informa actividad bacteriostática en todas las cepas estudiadas de la cual E. coli y S. agalactie parecen ser las más sensibles. Seguidos por P. aeuroginosa S. aureus y L. monoatogenes: siendo más evidente, pero aún presente, en C. albicans. Por los resultados obtenidos se concluye que el líquido folicular tiene una acción antibacteriana, probablemente selectiva y que el mantenimiento en ésta línea de investigación corroborará este hallazgo y posiblemente determinará los factores implicados en el mismo