scholarly journals Lactobacillus iners and gasseri, Prevotella bivia and HPV Belong to the Microbiological Signature Negatively Affecting Human Reproduction

2020 ◽  
Vol 9 (1) ◽  
pp. 39
Author(s):  
Giuseppina Campisciano ◽  
Valerio Iebba ◽  
Gabriella Zito ◽  
Stefania Luppi ◽  
Monica Martinelli ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Reproductive Tract ◽  
Culture Media ◽  
Abundant Species ◽  
Hpv Infection ◽  
Unexplained Infertility ◽  
Human Reproduction ◽  
Seminal Fluids ◽  
Infertile Couples ◽  
Follicular Fluids

Infertile couples undergoing the use of assisted reproductive technology are a good study model to evaluate the microbiological signatures affecting reproductive health. We tested vaginal lavages, follicular fluids, embryo culture mediums, and seminal fluids from 47 couples for their microbiome composition and HPV infection. Twenty-five infertile couples were diagnosed with unexplained infertility, whereas 22 were diagnosed with explained infertility. Lactobacilli were dominant in the vaginal lavages of both patient groups, and the most abundant species was L. iners (CST III), which is linked to a decreased fertility rate. Besides this, L. gasseri—which is known to be associated with oocyte DNA fragmentation and decreased sperm mobility—was identified in the seminal fluids, follicular fluids, and embryo culture media of the unexplained infertility group. Prevotella was increased in the seminal fluids of the explained infertility group, along with HPV-positive seminal fluids: an infection commonly associated with infertility, especially male infertility. Prevotella has been described to negatively affect sperm motility. Taken together, these results suggest that the profiling of the reproductive tract microbiome can add new perspectives to human reproduction.

Formulation of a complex serum-free medium (CSM) for use in the co-culture of mouse embryos with cells of the female reproductive tract

1991 ◽  
Vol 3 (1) ◽  
pp. 99 ◽  
Author(s):  
D Sakkas ◽  
AO Trounson
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Embryo Culture ◽  
Reproductive Tract ◽  
Culture Media ◽  
Mouse Embryos ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Cell Numbers

Co-culture of pre-implantation embryos with cells of the reproductive tract requires a medium that is beneficial to both embryos and cells. However, many studies in this area utilize media originally formulated for specific cell lines. In the present study, a complex serum-free medium (CSM) was formulated on the basis of the ionic compositions of existing embryo culture media and mouse oviductal fluid as well as the concentrations of growth factors that appear to benefit mouse embryo development. The study began by investigating the effect of altering the concentrations of K+ ions (0-40 mM) and sulfate ions (0-10 mM) in embryo culture media on the development of 2-cell mouse embryos. Mouse embryos showed improved cell numbers at the blastocyst stage when cultured in 10 mM K+ compared with Whittingham's T6 medium. Embryos were also cultured in T6 supplemented with bovine serum albumin (BSA) containing various concentrations of insulin, insulin-like growth factors I and II, fibroblast growth factor, and epidermal growth factor. Insulin concentrations of 100 ng mL-1 significantly (P less than 0.05) improved the cell numbers of 2-cell embryos cultured to the morulae and blastocyst stages compared with those cultured in T6 + BSA alone. CSM was formulated on the basis of the results of these experiments and was found to support both improved development of 2-cell mouse embryos and the culture of mouse fibroblast and mouse oviduct cells.

scholarly journals Impact of human papillomavirus infection on semen parameters and reproductive outcomes

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Hana Jaworek ◽  
Vladimira Koudelakova ◽  
Ivana Oborna ◽  
Blazena Zborilova ◽  
Jana Brezinova ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Sperm Count ◽  
Sperm Concentration ◽  
Hpv Infection ◽  
Human Reproduction ◽  
Semen Parameters ◽  
Reproductive Outcomes ◽  
Sperm Donors ◽  
Infertile Couples ◽  
Penile Swabs

Abstract Background Human papillomavirus (HPV) has been shown to adversely affect human reproduction. We aimed to evaluate the prevalence of human papillomavirus (HPV) infection in men and its correlation with semen parameters and reproductive outcomes. Methods Semen samples and penile swabs were collected from potential sperm donors (SD, n = 97) and male partners of infertile couples (IM, n = 328). The presence of HPV DNA in semen samples and penile swabs was analyzed. Associations between hrHPV positive status and fertility outcomes as well as socio-behavioral and health characteristics were evaluated using the R software package. Results High-risk HPV (hrHPV) genotypes were detected in 28.9% of SD and 35.1% of IM (P = 0.312). Penile swabs were more frequently positive for hrHPV genotypes than semen samples in both IM (32.3% vs. 11.9%, P < 0.001) and SD (26.8% vs. 6.2%, P = 0.006). Men with hrHPV positive semen samples had lower semen volume (median volume 2.5 ml vs. 3 ml, P = 0.009), sperm concentration (median concentration 16 × 106/ml vs. 31 × 106/ml, P = 0.009) and total sperm count (median count 46 × 106 vs. 82 × 106, P = 0.009) than men with hrHPV negative samples. No association was identified between penile hrHPV status and semen parameters. Conclusions Our findings indicate that penile HPV infection is common in both potential sperm donors and men from infertile couples. Although HPV positivity is higher in penile swabs, only HPV infection in semen samples affects sperm parameters. However, there was no association between hrHPV positivity in semen and fertility outcomes including abortion rate.

P-203 Applying artificial intelligence for ploidy prediction: The concentration of IL-6 in spent culture medium, blastocyst morphological grade and embryo morphokinetics as variables under consideration

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
B Aparicio Ruiz ◽  
L Bori ◽  
E Paya ◽  
M A Valera ◽  
A Quiñonero ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture Medium ◽  
Culture Media ◽  
General Description ◽  
Ann Model ◽  
Non Invasive ◽  
Morphokinetic Parameters ◽  
Testing Dataset ◽  
Standard Deviations ◽  
The Impact

Abstract Study question Would it be possible to predict embryo ploidy by taking into account conventional morphological and morphokinetic parameters together with IL-6 concentration in spent culture medium? Summary answer Our artificial neural network (ANN) trained with blastocyst morphology, embryo morphokinetics and IL-6 concentration distinguished between euploid/aneuploid embryos in 65% of the testing dataset. What is known already The analysis of spent embryo culture media represents the protein and metabolic state of the embryo and could be a non-invasive method of obtaining information about embryo quality. The impact of the presence/absence of several proteins in embryo culture samples over clinical results has been widely studied. The IL-6 is one of the most mentioned protein for its effect on embryo development, implantation and likelihood of achieving a live birth. In this initial attempt, we examined the predictive value for euploidy of a model that took into account the concentration of IL-6 in the spent culture medium. Study design, size, duration This prospective study included 319 embryos with PGT-A results. Out of the total, 127 were euploid and 192 aneuploid embryos. Concentration of IL-6 in spent embryo culture media (collected on the day of trophectoderm biopsy-fifth/sixth day of development), morphokinetic parameters (division time to 2 cells-t2; to 3 cells-t3, to 4 cells-t4; to 5 cells-t5 and time of blastocyst formation-tB) and blastocyst morphological grade (according to ASEBIR criteria) were considered to predict the embryo ploidy. Participants/materials, setting, methods Embryos were cultured in EmbryoScope. The chromosome analysis was performed using next-generation sequence technology. The concentration of IL-6 was measured in 20µL of spent embryo culture media with ELISA kits. Morphokinetic parameters were automatically annotated and the blastocyst morphology was evaluated by senior embryologists based on blastocele expansion, inner cell mass and trophectoderm quality. All the embryos were divided into 70% for training, 15% for validating and 15% for testing our ANN model with MatLab®. Main results and the role of chance The general description for the euploid embryo population was the following: 2% of the embryos were graded as A, 71% were graded as B and 28% were graded as C; the means and standard deviations were 25.32±2.97 hours (h) for t2, 35.33±5.15h for t3, 37.30±5.43h for t4, 48.24±6.62h for t5 and 103.93±12.8h for tB; and the average of IL-6 concentration was 1.51±0.70 pg/ml. The general description for the aneuploid embryo population was the following: 1% of the embryos were graded as A, 48% were graded as B and 51% were graded as C; the means and standard deviations were 26.13±3.51h for t2, 36.70±4.29h for t3, 38.20±4.24h for t4, 49.86±6.89h for t5 and 107.10±8.29h for tB; and the average of IL-6 concentration was 1.47±0.71 pg/ml. Our ANN model showed a higher general success rate as we increased the variables considered in the final prediction of euploid embryos. The accuracy, sensitivity and specificity for the testing dataset were: 0.60, 0.12 and 0.87 with morphokinetic parameters; 0.63, 0.24 and 0.93 with morphokinetics and IL-6 concentration; and 0.65, 0.16 and 0.96 with morphokinetics, IL-6 concentration and blastocyst morphological grade. Limitations, reasons for caution The low sensitivity and high specificity achieved in our models indicated that they were more capable of detecting aneuploid than euploid embryos. As this was a preliminary study, the small number of embryos included in the test (n = 48) was also a limitation. Wider implications of the findings The results showed that our model tended to classify the embryos as aneuploid. More euploid embryos would be necessary to train our model and achieve better results in the prediction of chromosomally normal embryos. Further studies with large number of embryos and additional variables could improve the non-invasive ploidy prediction. Trial registration number not applicable

P–582 High level of concordance between invasive and non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) at day5 and day6–7

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Biricik ◽  
V Bianchi ◽  
F Lecciso ◽  
M Surdo ◽  
M Manno ◽  
...  
Keyword(s):  
Data Analysis ◽  
Embryo Culture ◽  
Culture Media ◽  
Embryo Biopsy ◽  
Non Invasive ◽  
Culture Time ◽  
Ngs Data Analysis ◽  
High Level ◽  
Ngs Data

Abstract Study question To explore ploidy concordance between invasive and non-invasive PGTA (niPGT-A) at different embryo culture time. Summary answer High level (&gt;84%) of concordance rate for ploidy and sex, sensitivity (&gt;88%), and specificity (76%) were obtained for both day6/7 samples and day5 samples. What is known already The analysis of embryo cell free DNA (cfDNA) that are released into culture media during in vitro embryo development has the potential to evaluate embryo ploidy status. However, obtaining sufficient quality and quantity of cfDNA is essential to achieve interpretable results for niPGT-A. More culture time is expected to be directly proportional to the release of more cfDNA. But embryo culture time is limited due to in-vitro embryo survival potential. Therefore, it is important to estimate the duration of the culture that will provide the maximum cfDNA that can be obtained without adversely affecting the development of the embryo. Study design, size, duration A total of 105 spent culture media (SCM) from day5-day7 blastocyst stage embryos have been included in this cohort study. The cfDNA of SCM samples were amplified and analyzed for niPGT-A by NGS analysis. The SCM samples were divided into 2 subgroups according the embryo culture hours (Day5 and Day6/7 group). The DNA concentration, informativity and euploidy results have then been compared with their corresponding embryos after trophectoderm biopsy (TE) and PGT-A analysis by NGS Participants/materials, setting, methods Embryos cultured until Day3 washed and cultured again in 20µl fresh culture media until embryo biopsy on Day5, 6, or 7. After biopsy SCM samples were immediately collected in PCR tubes and conserved at –20 °C until whole genome amplification by MALBAC® (Yicon Genomics). The TE and SCM samples were analyzed by next-generation sequencing (NGS) using Illumina MiSeq® System. NGS data analysis has been done by Bluefuse Multi Software 4.5 (Illumina) for SCM and TE samples Main results and the role of chance Only the SCM samples which have an embryo with a conclusive result were included in this cohort (n = 105). Overall 97.1% (102/105) of SCM samples gave a successful DNA amplification with a concentration ranging 32.4–128.5ng/µl. Non-informative (NI) results including a chaotic profile (&gt;5 chromosome aneuploidies) were observed in 17 samples, so 83.3%(85/102) of SCM samples were informative for NGS data analysis. Ploidy concordance rate with the corresponding TE biopsies (euploid vs euploid, aneuploid vs aneuploid) was 84.7% (72/85). Sensitivity and specificity were 92,8% and 76,7%, respectively with no significant difference for all parameters for day 6/7 samples compared with day 5 samples. The false-negative rate was 3.5% (3/85), and false-positive rate was 11.7% (10/85). Limitations, reasons for caution The sample size is relatively small. Larger prospective studies are needed. As this is a single-center study, the impact of the variations in embryo culture conditions can be underestimated. Maternal DNA contamination risk cannot be revealed in SCM, therefore the use of molecular markers would increase the reliability. Wider implications of the findings: Non-invasive analysis of embryo cfDNA analyzed in spent culture media demonstrates high concordance with TE biopsy results in both early and late culture time. A non-invasive approach for aneuploidy screening offers important advantages such as avoiding invasive embryo biopsy and decreased cost, potentially increasing accessibility for a wider patient population. Trial registration number Not applicable

P–227 Fatty acid regulation of Nrf2/Keap1 pathway during mouse preimplantation embryo development

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
G Dionne ◽  
A J Watson ◽  
D H Betts ◽  
B A Rafea
Keyword(s):  
Oleic Acid ◽  
Fatty Acid ◽  
Palmitic Acid ◽  
Embryo Development ◽  
Embryo Culture ◽  
Preimplantation Embryo ◽  
Culture Media ◽  
Control Group ◽  
Blastocyst Development ◽  
Preimplantation Embryo Development

Abstract Study question Our objective is determining whether supplementing embryo culture media with palmitic acid and/or oleic acid impacts Nrf2/Keap1 antioxidant response pathways during preimplantation mouse embryo development. Summary answer Supplementation of embryo culture media with palmitic acid increases cellular Nrf2 levels per embryo after 48-hour culture, while oleic acid reverses this effect. What is known already Obese women experience higher incidence of infertility than women with healthy BMIs. The obese reproductive tract environment supporting preimplantation embryo development is likely to include enhanced free fatty acid (FFA) levels and increased accumulation of reactive oxygen species. Exposure to palmitic acid (PA) in vitro significantly impairs mouse embryo development while increasing ER stress mRNAs. Oleic acid (OA) reverses these effects. To further define effects of FFA exposure, we are characterizing the influence of FFAs on the Nrf2–Keap1 pathway and its downstream antioxidant defense systems. We hypothesize that PA treatment induces Nrf2-Keap1 activity, while OA treatment alleviates pathway activity. Study design, size, duration Female CD–1 mice (4–6 weeks) were super-ovulated via intraperitoneal injections of PMSG, followed 48 hours later by hCG. Female mice were mated with male CD–1 mice (6–8 months) overnight. Females were euthanized using CO2 and two-cell embryos were collected by flushing oviducts. Two-cell embryos were placed into KSOMaa-based treatment groups: 1) BSA (control); 2) 100µM PA; 3) 100µM OA; 4) 100µM PA+OA, and cultured for 48 hours (37 °C; 5% O2, 5% CO2, 90% N2). Participants/materials, setting, methods After 48-hour embryo culture, developmental stages of all mouse embryos were recorded. Immunofluorescence analysis of Nrf2 and Keap1 localization was performed for embryo treatments (BSA, 100µM PA, 100µM OA & 100µM PA+OA) using rabbit polyclonal anti-Nrf2 antibody, with Rhodamine-Phalloidin and DAPI staining. Embryos were imaged using confocal microscopy and Nrf2-positive cells were counted using ImageJ. Nrf2 and Keap1 mRNA abundances were assessed after culture in each treatment condition using RT-qPCR and the delta-delta Ct method. Main results and the role of chance Inclusion of 100µM PA in embryo culture significantly decreased blastocyst development frequency from 70.06±16.38% in the BSA (control) group to 11.61±8.19% in the PA-treated group (p &lt; 0.0001). Embryo culture with 100µM OA and 100µM PA+OA co-treatment did not significantly impair blastocyst development (OA: 61.59±8.07%, p = 0.4053; PA+OA: 63.53±7.63%, p = 0.6204). Embryo culture with PA treatment significantly increased the mean percentage of Nrf2-positive cells to 56.83±30.49% compared with 21.22±15.63% in the control group (p &lt; 0.0001). Conversely, 100µM OA and 100µM PA+OA treatments did not significantly affect Nrf2-positive cell frequencies compared with the control group (OA: 33.28±21.83%, p = 0.1825; PA+OA: 34.84±12.66%, p = 0.0691). Immunofluorescence results show that treating embryos with 100µM PA for 48 hours results in increased levels of cellular Nrf2, while combining 100µM PA with 100µM OA reversed these effects. Preliminary qPCR analysis showed no significant differences in Nrf2 or Keap1 relative transcript abundance between any embryo treatment groups. Nrf2 and Keap1 mRNA levels were both higher after embryo culture with 100µM OA than all other culture groups (p = 0.6268; p = 0.3201). Notably, Keap1 relative transcript levels dropped to undetectable levels after culture with 100µM PA, which suggests an increase in Nrf2 activation.Limitations, reasons for caution: While immunofluorescence localization of Nrf2/Keap1 provides insight into how the proteins behave during preimplantation embryo development, confocal images cannot determine protein-protein interactions or activity levels. Similarly, transcript information from RT-qPCR analysis only provides information about Nrf2 and Keap1 at the transcript level. Nrf2 activity will be assessed via downstream targets. Wider implications of the findings: The Nrf2–Keap1 pathway coordinates numerous cellular defence mechanisms, and is implicated in various diseases, including cancer. Establishing an impact of free fatty acid exposure on Nrf2–Keap1 during preimplantation embryo development will provide valuable information regarding the effects of maternal obesity on outcomes for embryos produced from these patients. Trial registration number Not applicable

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