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.\ud \ud 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: \ud (1) ‘colonizers’ if microorganisms were detected within the follicular fluid, but not within the vaginal swab (at the time of oocyte retrieval); or \ud (2) ‘contaminants’ if microorganisms detected in the vagina at the time of oocyte retrieval were also detected within the follicular fluid. \ud \ud 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

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 12 h of in vitro incubation. Follicular fluid bacteria may result in poor quality oocytes and/or embryos, leading to poor IVF outcomes

Number of women with colonized or contaminated follicular fluid and the causes of infertility.

1<p>‘Fertile’ women with infertile male partners.</p

Heat map key to shading based on microbial composition within the sample.

<p>Heat map key to shading based on microbial composition within the sample.</p

Heat map depicting IVF outcomes for fertile and infertile women with microorganisms detected from genital tract secretions during IVF cycles.

<p>Standard microbiological culture techniques were used to isolate and identify resident microorganisms from clinical specimens obtained from 262 women. Data represents the results obtained from culturing 262 vaginal swabs and 462 follicular fluids. All specimens were cultured on/in a range of microbiological culture media and broth. The heat map consists of a graphical two-dimensional matrix of colored squares, where each square represents the combined observations of the microorganisms isolated from all samples in each etiological cohort. The color of the square is dependent on the composition of the microorganisms at each anatomical site (FFL – left follicular fluid, FFR – right follicular fluid, VS – vaginal swab). Dark green squares represent specimens where only <i>Lactobacillus</i> spp. were isolated, light green squares represent specimens elaborating lactobacilli plus aerobic bacteria and/or microaerophilic bacteria. Light orange squares were used when <i>Lactobacillis</i> spp. were isolated alongside aerobic bacteria and/or microaerophilic bacteria, and/or anaerobic bacteria. Dark orange shading was used when only aerobic bacteria and/or microaerophilic bacteria were present but lactobacilli were absent and finally, red shading was used only if anaerobic species were the sole microbial isolates from the clinical specimen. The squares were ordered firstly by additional categorical data including the cause of infertility, as well as whether the microorganisms were defined as colonizers or contaminants at each site, and secondly by the IVF outcomes for fertilization rate, embryo discard rate, embryo transfer rate, pregnancy rate and live birth rate on the two axes. The combination of the three datasets into the one matrix allowed the cause of infertility, the resident microorganisms and the IVF outcomes to be visually examined and related to one another. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059062#pone-0059062-g003" target="_blank">Figure 3A</a> represents the positive IVF outcomes and has an overall cooler appearance (green – light orange), whilst <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059062#pone-0059062-g003" target="_blank">Figure 3B</a> represents the negative IVF outcomes and has an overall warmer appearance, being predominantly dark orange to red. In black shaded squares, no relationship was evident between the microbial groupings, the cause of infertility, and the IVF outcomes.</p

The most prevalent microbial species detected within the left or right follicular fluid and vagina in women with colonized or contaminated follicular fluid.

<p><i>Lactobacillus</i> spp., <i>Actinomyces</i> spp. and <i>Propionibacterium</i> spp. were detected more often in the colonized left follicular fluids than in the contaminated left follicular fluids (p<0.01, p<0.05 and p<0.05 respectively). <i>Actinomyces</i> spp. and <i>Propionibacterium</i> spp. were identified more often in colonized than in contaminated follicular fluids (p<0.05), while <i>Peptostreptococcus</i> spp. were isolated more frequently from contaminated follicular fluids from the right ovarian follicles (p<0.05). The vaginal swabs from women with colonized follicular fluid had a higher number of <i>Lactobacillus</i> spp. isolated (p<0.01) and also a higher number bacterial isolates when compared to women with contaminated follicular fluid specimens (p<0.05); however, the same species were detected in both groups of women.</p

The most prevalent microbial species detected from the left and right follicular fluids and vagina from women in this study.

<p>1 A–E The most prevalent microbial species detected from the left and right follicular fluids and vagina from women in this study are stratified based on the diagnosed cause of infertility. Bars represent one standard error.</p