Juan Antonio García Velasco

1. Microbiota y reproducción: ¿más
allá de la vulva y la vagina?
Dr. Juan A Garcia-Velasco
IVI Madrid

2. The Endometrial Factor

3. E 17 18 19 20 21 22 23
P P+4 P+5 P+6 P+7 P+8 P+9 P+10
Endometrial receptivity
Embryos attach ONLY under
optimal conditions

4. The Puzzle of the Endometrial Factor
Omics
Visuals
Ultrasound
Kasius et al., 2014
Histology
Noyes al., 1950
Coutifaris et al., 2004
Murray et al., 2007
Secretomics
Van der Gaast et al., 2002, 2009
Vilella et al., 2013
Transcriptomics
Riesewijk et al., 2003
Simon et al., 2005
Diaz-Gimeno et al., 2011, 2013
Aghajanova et al., 2012
Microbes
Microbiota
Franasiak et al., 2015
Moreno et al., 2016
Doppler
Kupesick et al., 2001
Omics
Hysteroscopy
Rambouts et al., 2016
Immunohistochemistry
Lessey et al., 1995
Kliman et al., 2006

5.  Dosage for COS (AMH, AFC, BMI, age)
 Type of protocol
 IVF vs ICSI
 Embryo number and quality
 ………
Personalized Medicine
However:
endometrial status at ET is treated equally
neglected customized approach
lack of knowledge?
Embryo transfer TIMING can be personalized

6. Technology helps

7. Transcriptome Proteome Metabolome
The age of –OMICS
Different subtypes of disease
TRANSCRIPTION TRANSDUCTION
Transcription
regulation
Alternative
splicing
Transduction
regulation
DNA RNA mRNA Protein METABOLITES

8. The Human Microbiome Project
• The human body contains 10-fold more
microbial cells than human cells
– Accounts for 1-3% of total body mass
– Some estimate as much as 2-6 lbs
• Termed the “second human genome”
– Linked in health & disease: diabetes, reumathoid
artritis, muscular dystrophy, multiple sclerosis, fibromyalgia,
and cancer
HMP, PLoS 2012
Wu, Nature Medicine 2009

9. hoy

10. Google: 6.900.000 hitsPubMed : 46,000 hits
2017

11. Techniques to study microbiota
Garcia-Velasco et al. RBMO 2017

12. P
Principal coordinate - 1
Principalcoordinate-2
Principal Coordinate Analysis

13. Human microbiome projectMicrobiota and disease
• Diabetes, CV…..

14. • Un microbioma saludable presenta baja diversidad de bacterias, siendo
rico fundamentalmente en lactobacilli.
• El paper de los Lactobacilli es proteger el epitelio vaginal fabricando una
capa gruesa que proteja la vagina de la infección por patógenos
(pH ~4.5)
Microbioma vaginal

15. Healthy women have a
low diverse vaginal
microbiota (few microbial
species present), mainly
lactobacilli
Women with unhealthy
vagina shows dysbiosis
(high number of
diverse microbial
species)
Vaginal Microbiota

16. Vínculo entre microbiota vaginal y fertilidad
1.- Live birth Shannon Diversity Index (SDI). Hyman et al J. Assist Reprod Genet 2012;29:105–115
2.- Moore DE et al. Fertil Steril 2000;74:1118–1124
3.- Sirota et al. Semin Reprod Med 2014;32:35–42
• Un microbioma rico en Lactobacilli y baja diversidad de
otras bacterias se asocia con buenos resultados en FIV 1,2
• Los lactobacillus que producen H2O2 tienen un impacto
positivo en la tasa de recién nacido vivo (88% vs 37%)2
• Durante un ciclo de FIV, 76% de la microbiota del tracto
reproductivo de las pacientes varía por los cambios
hormonales inducidos por el tratameinto

17. Vagina con
microbiota saludable
rico en Lactobacilli
Disbiosis puede
estar cuasada por
presencia de otras
especies
Esto tiene un
impacto en
fertilidad

18. Human microbiome projectVaginal microbiome of healthy
women
Ravel J et al. PNAS 2011; 108 (suppl 1): 4680-7
394 women
Heatmap of
log 10-transformed
microbial taxa found in
vaginal bacterial
communities of
394 women of
reproductive age

19. Human microbiome projectVaginal microbiome of healthy
women
Ravel J et al. PNAS 2011; 108 (suppl 1): 4680-7
Bacterial community groups – ethnic variations

20. Human microbiome projectLow lactobacilli and risk of
infection
• 5x higher risk of Chlamydia infection
• 3x higher risk of HPV
• 3x higher risk of HIV
Tamarelle J et al. Clin Microbiol Infect 2018; van de Wijgert J. Sex Transm Dis 2009

21. Human microbiome projectUteri is not sterile
Mitchell CM et al. Am J Obstet Gynecol 2015
UGT Vagina
L. inners 45% 61%
Prevotella spp 33% 76%
L. crispatus 33% 56%

22. Human microbiome projectUteri is not sterile
Mitchell CM et al. Am J Obstet Gynecol 2015

23. Human microbiome projectThe continuum of microbiota along
the female reproductive tract
Chen C et al. Nature Comm 2017

24. The stability of VAGINAL
microbiota
Kroon et al. Fertil Steril 2018

25. Endometrial Microbiome
Haahr et al. Hum Reprod 2016
Abnormal vaginal microbiota is associated with poor reproductive outcome

26. The relevance of UTERINE
microbiota
Simon C. Fertil Steril 2018

27. 35 INFERTILE PATIENTS UNDERGOING ART
IVF PATIENTS
Endometrial
fluid
MICROBIOTA
Healthy
Altered
Endometrial
biopsy
Receptive
Non-receptive
ERA TEST
ENDOMETRIAL MICROBIOTA & RECEPTIVITY
EMBRYO
TRANSFER
YES
NO
PREGNANCY
OUTCOME
PREGNANT
Non-pregnant
ENDOMETRIAL MICROBIOTA & IMPLANTATION
ONGOING
PREGNANCY
YES
NO
ENDOMETRIAL MICROBIOTA & PREGNANCY OUTCOME
Moreno et al. Am J Obstet Gynecol 2016
Endometrial Microbiome

28. COMPARISON OF THE AVERAGE BACTERIAL COMMUNITIES IN ENDOMETRIUM AND VAGINA MICROBIOTA OF FERTILE SUBJECTS
Only found in endometrial
samples of these women
EF= endometrial fluid
VA= vaginal aspirate
11
Endometrial Microbiome
Moreno et al. Am J Obstet Gynecol 2016

29. 1.00
0.00
0.75
0.50
0.25
Live birth No PregnantMiscarriage
LOW ABUNDANCE OF Lactobacillus IN ENDOMETRIAL MICROBIOTA IS ASSOCIATED WITH
POOR REPRODUCTIVE OUTCOMES IN IVF PATIENTS
Moreno et al., AJOG 2016 29
Moreno et al. Am J Obstet Gynecol 2016

30. Characteristics and Outcomes
LDM
(n=17)
NLDM
(n=15)
p-value
Age (y) 40.06±3.47 39.00±5.09 0.49
BMI (kg/m2
) 24.18±5.18 22.45±4.02 0.30
Previous pregnancies (n) 1.71±2.44 1.53±2.32 0.84
Previous miscarriages (n) 1.53±2.21 1.14±1.56 0.58
Metaphase II oocytes per cycle (n) 11.94±4.27 10.20±4.81 0.28
Fertilization rate per cycle 157/203 (77.34%) 118/153 (77.12%) 0.62
Transferred embryos per cycle (n) 1.65±0.49 1.73±0.59 0.65
Months between EF and transfer (n) 2.82±2.55 1.80±1.08 0.16
Pregnancy rate per transfer 12/17 (70.6%) 5/15 (33.3%) 0.03*
Implantation rate per transfer 17/28 (60.7%) 6/26 (23.1%) 0.02*
Ongoing pregnancy per transfer 10/17 (58.5%) 2/15 (13.3%) 0.02*
Miscarriage rates (%) 2/10 (16.7%) 3/5 (60.0%) 0.07
Live birth rate per transfer 10/17 (58.8%) 1§
/15 (6.7%) 0.002*
BMI: body mass index; LDM: Lactobacillus-dominated microbiota; NLDM: non-Lactobacillus-dominated microbiota;
*Chi Square (χ² test) and Student’s t-test were performed; *p-value<0.05; §: Voluntary termination of pregnancy.
LOW ABUNDANCE OF Lactobacillus IN ENDOMETRIAL MICROBIOTA IS ASSOCIATED WITH
POOR REPRODUCTIVE OUTCOMES IN IVF PATIENTS
31
Moreno et al. Am J Obstet Gynecol 2016

32. ¿Qué nos falta?
CONCLUSIONS:
First time to asses the effect of a well documented probiotic strain on vaginal microbioma and as well as its variations during
an OC cycle. L. rhamnosus CECT 8800 vaginal colonization and safety are shown: it is well-tolerated, increases significantly
Lactobacilli presence and has an influence in vaginal microbiota evolving it into healthier vaginotypes. All this may be useful
in IVF cycles.
Garcia-Velasco et al . ASRM 2018

33. Results
Microbiome evaluation by sequencing 16S rRNA
• Higher presence of L. rhamnosus after probiotic vs placebo
Garcia-Velasco et al . ASRM 2018

34. Results
• Probiotic microbiome at T3 & T4 clearly different from placebo: probiotic affects vaginal microbiome
T3 (end of COS)
T4 (3/4 after OPU)
Microbiome evaluation by sequencing 16S rRNA
Garcia-Velasco et al . ASRM 2018

35.  El factor endometrial es responsible del 10-20% de los
casos de fallo de implantación
 La microbiota vaginal y endometrial tiene una relevancia
hasta ahora desconocida en reproducción
 El porcentaje de Lactobacillus junto con patógenos
específicos pueden ayudar a predecir el resultado de un ciclo
 La valoración del factor endometrial puede expandirse de la
morfología al estudio molecular y/o microbiologia
Conclusiones

36. Gracias
juan.garcia.velasco@ivi.es