Field of the invention

The invention relates to the field of human reproduction, more in particular to situations in which human reproduction is failing. The present invention provides compositions for use in treatment promoting female fertility, establishing pregnancy or maintaining pregnancy in a subject in need thereof. Also described herein is a reliable and highly accurate method for predicting the chance that an assisted reproductive technology (ART) procedure, such as an in vitro fertilization and intra-cytoplasm ic sperm injection (ICSI) procedure will not lead to a successful pregnancy. Also provided are means and methods for predicting the chance that an assisted reproductive technology (ART) procedure, such as an in vitro fertilization and intra-cytoplasmic sperm injection (ICSI) procedure will lead to a successful pregnancy.

Background of the invention

Sub-fertility affects 10 to 15% of couples in the western world. This sub-fertility can in half of the cases be attributed to causes related to the female reproductive system, in 20-26% to the male and in 25-30% the cause is unknown (Evers, J.L., 2002, Lancet 360:151 -159). Many couples turn to an assisted reproductive technology (ART) procedure such as in vitro fertilization (IVF) or intra-cytoplasmatic sperm injection (ICSI) to fulfil their child-wish.

The success rate of these techniques is around 25% per started cycle (Andersen, A. et al. 2007, Hum. Reprod. 22:1513-1525 ). It would be of great emotional and economical benefit if this success rate could be improved.

Moreover, in view of the personal and societal burden of ART procedures, it is desirable to identify couples with a low chance for success very early on in the procedure, so that they can be offered alternative procedures to fulfill their child wish.

Thus both for improving the treatments and for deciding in individual cases whether to proceed there is a need for models that can accurately predict if a woman will not become pregnant and give live birth after IVF/ICSI.

For over a decade, models have been available that predict the chance of live birth on the basis of clinical data including age, number of previous failed IVF attempts and probable reason for infertility (Templeton, W. et al., 1996, Lancet 348:1402-1406). Nelson and Lawlor (Nelson, S.M. and Lawlor D.A. 201 1 , PLOS Medicine 8:1 -10) developed a model based on data from over 140.000 women, using stratification on age and cause of infertility, the procedure (to be) used, source of the egg and duration of the child wish. Selman et al., (J. Assisted Reproduction and Genetics 24: 395-399 (2007 )) discloses the detection of Lactobacillus and Staphylococcus in relation to IVF/pregnancy outcome. EP 2742359 B1 describes a method for predicting the chance of a successful or unsuccessful pregnancy in a subject, based on the relative amount of bacteria belonging to the group of lactobacillaceae and bacteria belonging to a species of Staphylococcus in a urine or vaginal sample.

Nevertheless, there remains a need for better, more reliable and easy-to-use methods for limiting the number of unnecessary ART procedures and predicting the chance of an unsuccessful outcome of an ART procedure. This would help to reduce costs in healthcare. Moreover, women with a child wish could be directed to alternative solutions earlier.

Summary of the invention

The invention relates to compostions for use in treatment promoting female fertility, establishing pregnancy or maintaining pregnancy in a subject wherein the compound is selected from a probiotic, a prebiotic or an antibiotic, or combinations thereof.

The invention relates to a method for predicting the likelihood that an assisted reproductive technology (ART) procedure will not result in a pregnancy, wherein a sample from a female mammalian subject taken before or during the ART procedure, is analyzed for at least one of the following parameters:

a) Presence of Gardnerella vaginalis,

b) Relative abundance of Lactobacillus species

c) Relative abundance of Lactobacillus jensenii,

d) Relative abundance of Proteobacteria and

wherein the subject has a high likelihood of not becoming pregnant as a result of the ART procedure,

I. if the sample comprises Gardnerella vaginalis or

II. if the relative abundance of Lactobacillus species is below a first

predetermined reference value, or

III. if the relative abundance of Lactobacillus jensenii is above a second

predetermined reference value or

IV. if the relative abundance of Proteobacteria is above a third predetermined reference value.

The invention also relates to a kit for performing a method according to the invention comprising a forward primer CTGGATCACCTCCTTTCTAWG (SEQ ID NO: 1 ) and a reverse primer AGGCATCCRCCATGCGCCCT (SEQ ID NO: 2) for the detection of an amplification product of Gardnerella vaginalis DNA wherein the Gardnerella vaginalis DNA amplification product has a length of 428 - 430 nucleotides, and wherein W denotes an A or a T and wherein R denotes an A or a G.

Detailed description of the invention

We provide herein a method for predicting the likelihood that an assisted reproductive technology (ART) procedure will not result in a pregnancy, wherein a sample from a female mammalian subject taken before or during the ART procedure, is analyzed for at least one of the following parameters:

a) Presence of Gardnerella vaginalis,

b) Relative abundance of Lactobacillus species

c) Relative abundance of Lactobacillus jensenii,

d) Relative abundance of Proteobacteria and

wherein the subject has a high likelihood of not becoming pregnant as a result of the ART procedure,

I. if the sample comprises Gardnerella vaginalis or

II. if the relative abundance of Lactobacillus species is below a first predetermined reference value, or

III. if the relative abundance of Lactobacillus jensenii is above a second predetermined reference value or

IV. if the relative abundance of Proteobacteria is above a third predetermined reference value.

In a preferred embodiment of the invention as described above, the presence of Gardnerella vaginalis is determined, as well as the relative abundance of Lactobacillus species, the relative abundance of Lactobacillus jensenii and the relative abundance of Proteobacteria.

In a further preferred embodiment, the invention relates to a method as described above, wherein the Gardnerella vaginalis is Gardnerella vaginalis IST1 . Gardnerella vaginalis IST1 is defined herein as a specific Gardnerella species that may be identified by performing vaginal microbial population analysis using amplification of the intergenic spaces (IS), according to the protocol provided by the manufacturer (IS-pro technique, IS-Diagnostics, Amsterdam, the Netherlands). IS-pro is an eubacterial technique based on the detection and categorisation of the length of the 16S-23S rRNA gene IS region. The length of this IS region is specific for each microbial species.

Gardnerella vaginalis IST1 is hereby defined as a species of Gardnerella vaginalis that results in a specific IS-fragment with a length of 428, 429 or 430 nucleotides when primers according to SEQ ID NO: 1 and SEQ ID NO: 2 are used.

The term ART procedure is used herein to indicate an artificial reproductive technology. In particular, the term relates to in Vitro Fertilization (IVF), Intra Cytoplasmic Sperm Injection (ICSI) and Intra Uterine Insemination (IUI).

The term relative abundance is used to indicate a fraction of the total amount or number of bacteria in a sample. The fraction is either expressed as a percentage (%) or as a number between 0 and 1 .

The term“high likelihood” in respect of predicting the chance of the success or failure of an ART procedure, is used herein to indicate that the predicted success or failure rate is higher than in the general population of women undergoing an ART procedure. In particular, the likelihood of not becoming pregnant is referred to as “increased” if the subject has a higher than 65% chance of not becoming pregnant as a result of the ART procedure if the criteria for a negative prediction as described herein are fulfilled. Higher than 65% in this respect includes for instance higher than 77%, such as 88% or even 94%.

Also, the likelihood of becoming pregnant is increased if the subject has a higher than 35% chance of becoming pregnant as a result of the ART procedure if the criteria for a positive prediction as described herein are fulfilled. Higher than 35% in this respect means 41 %, 49% or even 50% or more.

Gardnerella is a genus of Gram-variable-staining facultative anaerobic bacteria of which Gardnerella vaginalis is the only species. The organisms are small (1.0- 1 .5 pm in diameter) nonspore-forming, nonmotile coccobacilli. Once classified as

Haemophilus vaginalis and afterwards as Corynebacterium vaginalis, G. vaginalis grows as small, circular, convex, gray colonies on chocolate agar; it also grows on HBT agar. A selective medium for G. vaginalis is colistin-oxolinic acid blood agar. Determining the presence of Gardnerella vaginalis is preferably done by PCR, such as quantitative PCR.

Lactobacillus is a genus of Gram-positive, facultative anaerobic or microaerophilic, rod-shaped, non-spore-forming bacteria. They are a major part of the lactic acid bacteria group (i.e. they convert sugars to lactic acid). In humans, they constitute a significant component of the microbiota at a number of body sites, such as the digestive system, urinary system, and genital system. In women of European ancestry, Lactobacillus species are normally a major part of the vaginal microbiota. Lactobacillus forms biofilms in the vaginal and gut microbiota, allowing them to persist during harsh environmental conditions and maintain ample populations. Lactobacillus exhibits a mutualistic relationship with the human body as it protects the host against potential invasions by pathogens, and in turn, the host provides a source of nutrients.

The term Lactobacillus species is used herein to refer to all Lactobacillus species collectively.

Lactobacillus jensenii is a common inhabitant of the lower reproductive tract in healthy women. In a normal population, L. jensenii makes up to about 23% of vaginal microflora that is naturally occurring.

The Proteobacteria are a major phylum of bacteria. They are gram-negative bacteria. This means they do not retain the violet dye in the Gram staining protocol. In a Gram stain test, a counterstain (commonly safranin) is added after the crystal violet, colouring all gram-negative bacteria with a pink colour. The test itself is useful in classifying two distinct types of bacteria based on the structural differences of their cell walls.

Proteobacteria include a wide variety of pathogens, such as Escherichia coli, Salmonella, Vibrio, Helicobacter, and many other notable genera. Others are free-living, and include many of the bacteria responsible for nitrogen fixation. The group is defined primarily in terms of ribosomal RNA (rRNA) sequences.

There are numerous ways for determining these microorganisms and the skilled person is well aware of techniques on how to determine and quantify the relative amounts of Gardnerella, Lactobacillus species, L. jensenii and Proteobacteria in a sample. We provide herein the results of a study wherein we determined the presence and relative amounts of these bacteria in samples obtained from a population of 192 women undergoing an ART procedure.

We correlated the presence and relative amounts of these microorganisms and found that their presence and/or abundance was indicative of the success rate or failure rate of an ART procedure.

Out of 192 women, 125 did not become pregnant after the first attempt whereas 67 did become pregnant. This is a failure rate of the ART procedure of 65% and a success rate of 35% (Table 1 ).

In a method according to the invention, the relative abundance of a particular species or genus of bacteria has to be compared with a predetermined reference value or cut-off value. The predetermined reference value may be any suitable cut-off value. This process of determining a suitable cut-off value is well within the skills of a skilled person and can easily be determined empirically by the skilled person.

Preferably it is a value derived from the bacterial composition of samples obtained from a comparable population as the test population. Even more preferred is a reference value obtained from an average value of several independent experiments of ART procedures in a reference population. The skilled person is aware of the particulars of determining reference values for measuring and determining the relative abundance of bacteria.

Hence, the predetermined reference value may be empirically determined or arbitrarily chosen in order to achieve appropriate specificity and/or sensitivity of the method. A skilled person is fully aware how to choose an appropriate reference value. A skilled person will know how to alter the predetermined reference value in order to obtain the desired specificity and sensitivity of the method.

As an example, in the method described above, the first predetermined reference value may be between 15 and 25%, such as 20%, the second predetermined reference value may be between 25 and 45%, such as 35% and the third predetermined reference value may be between 18 and 38%, such as 28%.

When the first, second and third reference values were chosen as 20%, 35% and 28% respectively and the data from table 1 were combined with the data of the microbial composition of the vaginal bacterial population, it appeared that 32 of the 125 unsuccessful ART outcomes could be correctly predicted, i.e. 26% of the cases where the ART procedure failed, could be correctly predicted (Table 2).

Hence, a method according to the invention as disclosed above, produces highly reliable results, i.e. it failed to predict an unsuccessful outcome of the ART procedure in only 2 cases. In these two cases, the ART procedure resulted in a pregnancy which is considered the desired outcome; i.e. a successful outcome. The precision of the method to predict that a subject will not become pregnant, as described above, is therefore 32/34 = 94% (Table 5).

Table 2: Correlation matrix based on at least one out of 4 parameters

If the method according to the invention as described above would have been used as an exclusion criterion in this study, then 34 women would have been excluded from this study and 158 instead of 192 women would have been allowed into the procedure, of which 65 would have become pregnant. This means that the success rate of the ART procedure in that case would have been increased from 67/192 = 35% to 65/158= 41 %. This is an increase of the relative efficiency of the ART procedure with 6%. An additional advantage would be that the 34 women would not have to undergo an ART procedure or procedures before they would have been offered alternative approaches.

If applied to the present population in the study as described herein, the selection procedure would have led to a reduction of the number of ART procedures with 34/192 = 18%. The total average costs for an ART procedure such as IVF or ICSI are in the order of €5.000. In total, applying the method according to the invention would have saved on average€900 per IVF/ICSI patient, or in other terms, the costs of the procedure would have been reduced with 18%.

We also determined the predictive value of a method based on the presence of Gardnerella vaginalis, such as G. vaginalis 1ST, in particular G. vaginalis IST1 , alone. It appeared that women with Gardnerella vaginalis IST1 had an 88% chance of not becoming pregnant (Table 3, Table 5). Hence, a method as described above, wherein Garnerella vaginalis is G. vaginalis IST1 yielded good results.

Table 3: Correlation matrix based on Gardnerella vaginalis IST1

Hence, the invention also relates to a method as described above wherein the presence of Gardnerella vaginalis, preferably G. vaginalis 1ST, such as G. vaginalis IST1 is determined in the sample and wherein the subject has a high likelihood of not becoming pregnant as a result of the ART procedure if the sample comprises Gardnerella vaginalis, preferably G. vaginalis IST1 .

We also determined the presence of two Lactobacillus species, L. crispatus and L. iners in the samples provided, using the ISPRO technique (Example 4). The results are shown in Table 4.

Lactobacillus crispatus is a common inhabitant of the lower reproductive tract in healthy women. In a normal population, L. crispatus is the dominant species in more than 30% of all women of reproductive age.

Lactobacillus iners is also a species in the genus Lactobacillus. It is a Gram positive, catalase-negative, facultatively anaerobic rod-shaped bacterium. Lactobacillus iners is a normal inhabitant of the lower reproductive tract in healthy women. The genomes of at least 15 strains have been sequenced and encode between 1 ,152 and 1 ,506 proteins. Therewith this species has one of the smallest Lactobacillus genomes compared to other species, such as L. crispatus, which typically encodes more than twice as many proteins.

There are numerous ways for determining these microorganisms and the skilled person is well aware of techniques on how to determine and quantify the relative amounts of Lactobacillus species, L. crispatus and L. iners in a sample.

We found that these two species (L. crispatus and L. iners) were also indicative of the failure or success of an ART procedure.

In particular, it was found that a subject had a high likelihood of not becoming pregnant as a result of an ART procedure, if the relative abundance of Lactobacillus crispatus was above a fourth predetermined reference value. This fourth predetermined reference value was preferably chosen between 50 and 70%, such as 60%. When 60% was taken as the fourth reference value, 65 women fulfilled this criterion, of which 15 became pregnant as a result of the ART procedure (table 5). This is a failure rate of 77%, which is higher than the failure rate in the entire group (table 5).

It was also found that a subject had a high likelihood of becoming pregnant as a result of the ART procedure, if the relative abundance of Lactobacillus crispatus was below a fifth predetermined reference value. This fifth predetermined reference value was preferably chosen between 50 and 70%, such as 60%. When 60% was taken as the fifth reference value, 127 women fulfilled this criterion, of which 52 became pregnant as a result of the ART procedure (table 5). This is a success rate of 41 %, which is higher than the success rate in the entire group (table 5). These results are shown in table 4 and table 5.

Table 4: Correlation matrix

We also determined the relative abundance of Lactobacillus iners and found that the subject had a high likelihood of becoming pregnant as a result of the ART procedure, if the relative abundance of Lactobacillus iners was above a sixth

predetermined reference value. This sixth predetermined reference value was preferably chosen between 50 and 70%, such as 60%. When 60% was taken as the sixth reference value, 38 women fulfilled this criterion, of which 19 became pregnant as a result of the ART procedure (table 5). This is a success rate of 50%, which is higher than the success rate in the entire group (table 5).

Hence, we describe a method for predicting the likelihood that an assisted reproductive technology (ART) procedure will not result in a pregnancy, wherein a sample from a female mammalian subject taken before or during the ART procedure, is analyzed for the relative abundance of Lactobacillus crispatus and wherein the subject has high likelihood of not becoming pregnant as a result of the ART procedure, if the relative abundance of Lactobacillus crispatus is above a fourth predetermined reference value.

We also describe a method for predicting the likelihood that an assisted reproductive technology (ART) procedure will result in a pregnancy, wherein a sample from a female mammalian subject taken before or during the ART procedure, is analyzed for the relative abundance of Lactobacillus crispatus and wherein the subject has high likelihood of becoming pregnant as a result of the ART procedure, if the relative abundance of Lactobacillus crispatus is below a fifth predetermined reference value .

We also describe a method for predicting the likelihood that an assisted reproductive technology (ART) procedure will result in a pregnancy, wherein a sample from a female mammalian subject taken before or during the ART procedure, is analyzed for the relative abundance of Lactobacillus iners and wherein the subject has high likelihood of becoming pregnant as a result of the ART procedure, if the relative abundance of Lactobacillus iners is below a sixth predetermined reference value .

We also describe herein a method for predicting the likelihood that an assisted reproductive technology (ART) procedure will result in a pregnancy, wherein a sample from a female mammalian subject taken before or during the ART procedure, is analyzed for the presence of Lactobacillus crispatus and of Lactobacillus iners, and wherein the relative amounts of L. crispatus [LC] and L. iners [LI] are determined, and wherein the likelihood of a pregnancy is increased if [LC] is below a seventh predetermined reference value and wherein

a. [LC} < (a * [LI]) + b and

b. [LC} > (c * [LI]) + d and

wherein a is a value between -0.55 and -0.70, b is a value between 0.80 and 0.90, c is a value between -0.50 and -0.65 and wherein d is a value between 0.3 and 0.45.

In a preferred embodiment, a = -0.62, b = 0.85, c = -0.58 and d = 0.38. In this case, 77 of the 192 women from the study described herein were found to fulfill the criterion, of which 38 (49%) became pregnant as a result of the ART procedure (Table 5). These results are graphically represented in figure 1 .

Particularly good results were obtained when the fourth, fifth, sixth and seventh predetermined reference values were independently from each other chosen between 50% and 70%, even more in particular 60%.

We also describe herein compositions for use in treatment aimed at promoting female fertility, establishing pregnancy or maintaining pregnancy in a subject. The compound is compound is selected from a probiotic, an antibiotic, a prebiotic, or combinations thereof.

It was found that the compounds, when provided to a subject, can enhance the chance that any pregnancy attempt, including the use of an assisted reproductive technology (ART) procedure, such as in vitro fertilization and intra-cytoplasmic sperm injection (ICSI) procedure will more likely lead to a successful pregnancy.

It was realized by the current inventors, based on the analysis of the vaginal microbiome prior to the start of an ART procedure, and as disclosed herein, that the relative amounts (or abundance) of bacterial species, in particular Lactobacillus crispatus, Lactobacillus iners, Lactobacilles jensenii, Proteobacteria and Gardnerella vaginalis IST1 were associated with pregnancy rates (Table 5). In particular, a relative abundance of L. iners of >50%, preferably >60%, a relative abundance of L. crispatus <70%, preferably <60%, a relative abundance of Proteobacteria <40%, preferably <28%, non-detectable (very low) levels of Garnerealla vaginalis IST1 and a relative abundance of L. jensenii <40%, preferably <35% were associated with enhanced pregnancy rates. Based thereupon it was realized that any treatment of a subject with the aim of modulating the vaginal microbiome of said subject towards or into a (more) pregnancy- competent status may promote female fertility or establishing pregnancy or maintaining pregnancy. Such treatment would, for example, aim towards optimally balanced L.

crispatus and L. iners levels, and, for example, low levels of G. vaginalis IST1 and Proteobacteria.

In some embodiments, there is provided for a composition for use in treatment as disclosed herein wherein the composition comprises a probiotic and wherein the probiotic is selected from the group consisting of Lactobacilli, Bifidobacteria, Streptococci, Lactococci and Saccharomyces, preferably wherein the probiotic is Lactobacillus iners, Lactobacillus rhamnosus, preferably Lactobacillus rhamnosus GR-1 , or Lactobacillus reuteri, preferably Lactobacillus reuteri RC-14, or combinations of two or three thereof.

The at least one probiotic microorganism is preferably selected from the group of Lactobacilli, Bifidobacteria, Streptococci, Lactococci and Saccharomyces. For example, the probiotic organism may be a lactic acid bacteria, bifidobacteria and bacilli. Non-limiting examples include Bacillus coagulans, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fortis, Lactobacillus johnsonii, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Saccharomyces cerevisiae (boulardii) and combinations thereof.

Preferred probiotic bacteria comprise Lactobacillus rhamnosus, preferably Lactobacillus rhamnosus GR-1 and Lactobacillus, preferably Lactobacillus reuteri RC-14. Even more prefererred is a combination of Lactobacillus rhamnosus GR-1 and

Lactobacillus, preferably Lactobacillus reuteri RC-14. Such probiotic bacteria, or combination of bacteria have been shown in the art to modify the vaginal microbiota, even when provided as an oral supplement, for example thereby providing an increased relative abundance of L. iners (Macklaim et al (2015) Microbial Ecology in Health and Disease, 26:1 , 27799).

In a particular preferred embodiment the subject is orally treated with L.

rhamnosus GR-1 and L. reuteri RC-14, for example for a period Of 20 - 40 days, e.g. 28 days.

In another preferred embodiment the subject is treated with a the antibiotic fluconazole, for example as a single dose of 100 - 200 mg, preferably 150 mg.

In another preferred embodiment the subject is treated with a single dose of fluconazole in combination with oral with L. rhamnosus GR-1 and L. reuteri RC-14, for example for a period Of 20 - 40 days, e.g. 28 days. In some embodiments at least 1 to 4 10 ⁹ , for example at least 2,7 10 ⁹ of each of the probiotic bacteria are provided per day.

According to another embodiment an effective amount of the bacteria that were indentified herein as benefical are provided to the subject. For example the subject can be provided with an effective amount (for example a dose of each bacteria from 10 ³ to about 10 ¹³ CFU/ml or equivalent thereof) of, in a preferred embodiment, L. iners. By providing beneficial (probiotic) bacteria to the subject the relative abundance of said bacteria will be increased. Preferably the relative levels (abundance) of L. liners should be increased to >60%, for example prior an ART procedure.

The beneficial bacteria may be provided directly into the vagina, or by any other route, including oral. Modulation of the vaginal microbiome through administration of bacterial strains is possible, as has been shown by Burton et al (AEM (2003) 69:97-101 ).

In some embodiments, there is provided that in addition to the treatment with an probiotic, the treatment furhter comprises treatment of the subject with an antibiotic, preferably wherein the antibiotic is selected from the group consisting of tinidazole, fluconazole, metronidazole, clindamycin and secnidazole, or combinations thereof.

In some embodiments there is provided for a composition for use in treatment as disclosed herein wherein the composition comprises an antibiotic and wherein the antibiotic is selected from the group consisting of azoles, penicilins, cephalosporins, clindamycin, vancomycin, tetracyclines, chlorampheicol, trimethoprim- sulfamethoxazole, erythromycins, glycopeptides, sulfa drugs, iodine and chlorhexidine and mixtures thereof, prefereably wherein the antibiotic is selected from the group consisting of tinidazole, fluconazole, metronidazole, clindamycin and secnidazole, or combinations thereof.

By using specific antibiotics inhibition or even eradication of non-beneficial bacteria may be obtained. For example inhibition of L. crispatus, G. vaginalis IST1 and Proteobacteria can be achieved using specific antibiotics/antimicrobials. For example, adverse bacteria (such as L. crispatus, G. vaginalis and proteobacteria) can be inhibited by application of specific antibiotics that will decrease these species while increasing the relative abundance of the beneficial bacteria (L. iners).

Antibiotics may, for example, be selected from the group consisting of Azoles (including triazoles such as Fluconazole) Penicilins, Cephalosporins, Clindamycin, Vancomycin, Tetracyclines, Chlorampheicol, Trimethoprim-sulfamethoxazole,

Erythromycins, Glycopeptides, Metronidazole, Sulfa drugs, Iodine and chlorhexidine and mixtures thereof. Particular non-limiting examples include fluconazole, tinidazole, metronidazole, clindamycin or secnidazole. For example, oral tinidazole has been shown to inhibit G. vaginalis IST1 .

In some embodiments the composition for use in treatment as disclosed herein comprises a prebiotic and wherein the prebiotic is selected from the group consisting of lactate, lactulose, galacto-oligosaccharides (GOS), fructo-oligosaccharides (FOS), fiber gums, inulins, isomalto-oligosaccharides, lactilol, lactosucrose, oligofructose, pyrodextrins, soy oligosaccharides, transgalacto-oligosaccharides (TOS), and xylo- oligosaccharide, or combinations thereof.

The skilled person is well aware of prebiotics. Prebiotics have been defined by the International Scientific Association for Probiotics and Prebiotics (ISAPP) as“a substrate that is selectively used by a host microorganism to produce a health benefit”.

Prebiotics are, for example, non-digestible food ingredients which stimulate the growth and activity of beneficial microorganisms, mainly bifidobacteria and lactobacilli.

Prebiotics mainly consist of oligosaccharides, sugar molecules of three to six chains and soluble fiber.

Non-limiting examples of such prebiotics include lactate, specific sugars, e.g. lactulose, galacto-oligosaccharides (GOS) or fructo-oligosaccharides (FOS). Such probiotics have been shown to stimulate growth of particular Lactobacilli (Collins et al, (2018, AEM 84:e2200). Other examples of oligosaccharides which exhibit prebiotics characteristics are, for instance, fiber gums, inulins, isomalto-oligosaccharides, lactilol, lactosucrose, oligofructose, pyrodextrins, soy oligosaccharides, transgalacto- oligosaccharides (TOS), and xylo-oligosaccharide.

As will be understood by the skilled person, is some preferred embodiments, a combination of two or more prebiotics, or a combination of two or more prebiotics, or a combination of two or more antibiotics may be used.

In some embodiments there is provided for a composition for use in treatment as disclosed herein wherein in the treatment comprises the use of a probiotic and an antibiotic, a probiotic and a prebiotic, an antibiotic and a prebiotic or a probiotic, an antibiotic and a prebiotic.

In other embodiments, combinations of at least two of a prebiotic, a probiotic and an antibiotic are used. For example, antibiotics may be used to suppress an adverse microbiome in general in combination by providing beneficial bacteria (L. iners) to the subject and/or by providing prebiotics to the subject in order to increase the relative abundance of beneficial bacteria (L. iners). It has for example been shown that a combination of an antibiotic (tinidazole, or fluconazole) together with a combination of two probiotics, Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 increases the relative abundance of L. iners in the vagina. Other combinations include combinations of probiotics and prebiotics, probiotics and antibiotics, prebiotics and antibiotics, and probiotics, antibiotics and probiotics.

Also provided is a composition for use in treatment according to any of the preceding claims wherein the treatment further comprises an assisted reproductive technology (ART) procedure, preferably wherein the ART procedure is an in vitro fertilization (IVF) procedure, preferably an intra-cytoplasm ic sperm injection (ICSI) procedure. In such embodiment, the subject can be treated with the compounds as disclosed herein prior to the ART procedure in order to provide for an improved vaginal microbiota. For example, the subject may be treated for a period of at least one day, week or month before an ART procedure is initiated. In some embodiment the subject is provided with the compounds also during and after the ART procedure has been performed (the subject has been treated with the ART procedure).

In a preferred embodiment there is provided for composition for use in treatment as disclosed herein wherein the composition comprises:

a. Lactobacillus rhamnosus GR-1 , and wherein the treatment preferably further comprises treatment with Lactobacillus reuteri RC-14 or with an antibiotic selected from the group consisting of tinidazole and fluconazole, or with Lactobacillus reuteri RC-14 and an antibiotic selected from the group consisting of tinidazole and fluconazole;

b. Lactobacillus reuteri RC-14, and wherein the treatment preferably

further comprises treatment with Lactobacillus rhamnosus GR-1 or with an antibiotic selected from the group consisting of tinidazole and fluconazole, or with Lactobacillus rhamnosus GR-1 and an antibiotic selected from the group consisting of tinidazole and fluconazole; or c. An antibiotic selected from the group consisting of tinidazole and

fluconazole, and wherein the treatment preferably further comprises treatment with Lactobacillus rhamnosus GR-1 , Lactobacillus reuteri RC- 14 or with Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC- 14.

In some embodiments, the composition for use in treatment as disclosd herein is used in the treatment of a subject that increases the relative abundance of Lactobacillus iners, preferably to more than 50%, more preferably to more than 60%, decreases the relative abundance of Lactobacillus crispatus, preferably to less than 70%, more preferably to less than 60%, decreases the relative abundance of proteobacteria, preferably to less than 40%, more preferably to less than 28%, decreases the relative abundance of Lactobacillus jensenii, preferably to less than 40%, more preferably to less than 35%, and/or decreases Garnerealla vaginalis IST1 to less than the detection limits of current standard Polymerase Chain Reaction (PCR) and or next generation sequencing (NGS), preferably wherein Garnerealla vaginalis IST1 is eradicated, in the vaginal microbiome of the the subject.

Also provided is for the composition for use in treatment as disclosed herein wherein the subject is a subject that has a high likelihood of not becoming pregnant, preferably as determined with a method for predicting the likelihood that an assisted reproductive technology (ART) procedure will not result in a pregnancy.

Indeed, in some embodiments relating to the compostions for use in treatment the subject is a subject that has a high likelihood of not becoming pregnant, preferably as determined with the method for predicting the likelihood that an assisted reproductive technology (ART) procedure will not result in a pregnancy as described herein in detail. In particular such subject may benefit from the treatments as disclosed herein.

Also provided is for a composition for use in treatment as disclosed herein wherein the treatment is for a period of at least one day, week or month, preferably wherein the treatment is started for a period of at least one day, week or month before an ART procedure is initiated.

As will be understood by the skilled person the compounds for use in the treatment may be provided to the subject by any suitable route of administration, including but not limited to administration: systemic, oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral or mucosal application. Preferably administration is orally or vaginally.

The compounds may be applied with or without a pharmaceutically acceptable carrier. Dosage forms include but are not limited to a suppository douche, liquid, tablet, capsule, (adhesive) patch, cream paste, (sanitary) pad, liner, interlabial device, wipe, pessary, tampon, pantiliner, capsule, cream, paste, drink, gum, mouth wash, mouth spray or other mucoadhesive medium, nasal spray, nasal packing, and any other type of administration form. In combinations of the compounds as disclosed herein, the compounds may be provided simultaneously to the subject in the same dosage form, but may also be provided to the subject in sequential order, either using the same route of administration or different. The compounds may be provided to the subject once or repeated over a longer period of time, e.g. days or weeks.

Also provided is for the use of a probiotic, a prebiotic or a antibiotic, or combinations thereof for promoting female fertility, establishing pregnancy or maintaining pregnancy in a subject, with the provisio that the use of said probiotic, prebiotic or a antibiotic, or combinations thereof, is not for therapeutic treatment. For example, the probiotic, prebiotic and antibiotic may be taken as a dietary supplement or the like on order to maintain an healthy microbiota in the vagina of a women.

Also disclosed are methods of treatment of a subject using the compounds as disclosed herein. All embodiment and preferences described herein in the context of the compostion for use in the treatment likewise apply for embodiments pertaining to the methods of treatment and for the use of a probiotic, a prebiotic or a antibiotic, or combinations thereof for promoting female fertility, establishing pregnancy or maintaining pregnancy in a subject, with the provisio that the use of said probiotic, prebiotic or a antibiotic, or combinations thereof, is not for therapeutic treatment.

Legend to the figure

Figure 1 : Scatter plot of data obtained with one of the methods exemplified herein wherein the likelihood of a pregnancy is increased if [LC] is below 60% and wherein

[LC} < (a * [LI]) + b and wherein

[LC} > (c * [LI]) + d and wherein

a = -0.62,

b = 0.85,

c = -0.58 and

d = 0.38.

Table 5

Examples

Example 1 : Study population

This prospective study of the vaginal microbiome of sub-fertile women of reproductive age was carried out in eight IVF centres in the Netherlands. The participating centres were: Erasmus Medical Centre (Rotterdam), Radboud UMC (Nijmegen), UMC Utrecht (Utrecht), VU MC (Amsterdam), Isala kliniek (Zwolle), Sint Elisabeth Ziekenhuis (Tilburg), MC Kinderwens (Leiderdorp), MUMC+ (Maastricht). Inclusions took place over an almost one year period (1 st June 2015 to 31 st March 2016). The protocol was approved by the Institutional Review Board of the Erasmus University Medical Centre. Written informed consent was obtained from all participants.

Women who visited the outpatient clinic of reproductive health clinic and who were expected to undergo their first IVF (with or without ICSI) procedure within two months were approached to participate in this study. Criteria to be fulfilled were: women between the ages of 20 and 44 years and having a male partner. Those excluded from the study were: women with an indication for emergency IVF because of cancer or other reasons, endometriosis AFS 11 I/I V and pretreated with a Gonadotrophin-releasing hormone (GnRH) analogue, use of hormonal contraceptives 3 months prior to start IVF or IVF-ICSI (exclusive 3 weeks use of oral contraceptive pill for the purpose of cycle regulation) and who had a previous pregnancy or miscarriage in medical history.

In this study, 301 women were enrolled at first. Twenty-one patients were excluded on the basis of the exclusion criteria, another 86 left the study for personal or unknown reasons. Two samples were lost due to handling errors, hence the study was eventually conducted with samples from 192 individuals.

Example 2: Materials

Participants obtained a vaginal swab by themselves prior to the start of the IVF or IVF-ICSI procedure. A self-collecting method was chosen, because it is minimal invasive for the patient and therefore suitable for use in the daily practice. The vaginal samples were taken with FLOQSwabs™ (Copan Italia S.p.A., Italy) and the participants were instructed to insert the swab 3-5 centimetre into the vagina, then to rub the swab along the vaginal wall for 10-15 seconds. After this procedure the swabs were

immediately placed in Eppendorf tubes filled with reduced transport fluid (RTF) buffer, obtained from IS-Diagnostics (IS-Diagnostics, Amsterdam, the Netherlands). Up to the analysis, the samples were stored at -20 to -80 °C degrees in the freezer.

Urine samples were collected in a sterile urine collecting device of 100 ml. A 10 ml sample was centrifuged for 10 minutes at 1500 RCF. The supernatant was decanted and the pellet was re-suspended in 3 ml urine. The re-suspended sample was stored for further processing at -20 degrees Celsius.

Samples were transported on dry ice from the 8 clinics to the microbiological laboratory of IS-Diagnostics, where the analyses were performed.

Example 3: DNA isolation

DNA extraction was performed from the vaginal swabs with the Chemagen (Chemagen, Baesweiler, Germany) automated DNA extraction machine using the buccal swab extraction kit according to the manufacturer’s instructions. First the swabs were thawed and vortexed. 200 mI of sample was incubated with 200 mI Chemagen lysisbuffer and 10 mI Proteinase K (Qiagen, Hilden, Germany) at 56 degrees Celsius while shaking at 500 rpm. DNA was extracted using the protocol buccal Swab Prefilling. Elution of DNA was in 100mI of Chemagen Elution buffer.

DNA was extracted from concentrated urine suspensions with the Chemagen (Perkin-Elmer, Baesweiler, Germany) automated DNA extraction machine using the buccal swab extraction kit according to the manufacturer’s instructions. In short, urine samples were thawed and vortexed. 200 mI of sample was incubated with 200 mI

Chemagen lysis buffer and 10 mI Proteinase K at 56 degrees Celsius while shaking at 500 rpm. Elution of DNA was in 100mI of Chemagen Elution buffer.

Example 4: Interspace (IS) profiling

Amplification of the intergenic spaces (IS) regions was performed with the IS- pro assay, according to the protocol provided by the manufacturer (IS-Diagnostics, Amsterdam, the Netherlands). IS-pro is an eubacterial technique based on the detection and categorisation of the length of the 16S-23S rRNA gene IS region. The length of this IS region is specific for each microbial species. Phylum-specific fluorescently labelled PCR primers are used for taxonomic classification.

Briefly, the procedure consists of two separate standard PCRs: the first PCR mixture contains two different fluorescently labelled forward primers targeting different bacterial groups and three reverse primers providing universal coverage for those groups. The first forward primer is specific for the phyla Firmicutes, Actinobacteria, Fusobacteria, and Verrucomicrobia (FAFV), and the second labeled forward primer is specific for the phylum Bacteroidetes. A separate PCR with a labeled forward primer combined with seven reverse primers is specific for the phylum Proteobacteria [Budding, E. et al., J. Clin. Microbiol. (2016) 54: 934-943].

GeneAmp 9700 PCR system (Applied Biosystems, Foster City, CA) performed the amplications. After PCR, 5 mI of PCR product was mixed with 20 mI of formamide and 0.2 mI of custom size marker (IS-Diagnostics). DNA fragment analysis was performed on an ABI Prism 3500 genetic analyzer (Applied Biosystems). Data were analyzed with the IS-pro proprietary software suite (IS-Diagnostics), and the results are presented as microbial profiles. Automated species calling of IS-pro peaks was done with the dedicated IS-pro software suite (IS-Diagnostics), in which peaks are linked to a database containing IS-profile information of >500 microbial species. Peaks of <128 relative fluorescence units (RFU) were regarded as background noise and were discarded from further analysis. The whole procedure, from DNA isolation to analyzed data, was performed within 5 hours.

Example 5: Outcome measurement

Pregnancy outcome after the first embryo transfer (ET), was used as endpoint. Ongoing pregnancy was defined as a fetus with heart activity established with the use of an ultrasound between 7-9 weeks of gestation.

Example 6: Determination of Gardnerella vaginalis IST1

Gardnerella vaginalis IST1 was identified by performing vaginal microbial population analysis with the IS-pro technique. G.vaginalis IST1 was detected by presence of a specific IS-fragment with a length of 428-430 nucleotides.

Example 7: Determination of microbiome

Microbiome analysis was performed with the IS-pro technique as described previously [Automated Broad-Range Molecular Detection of Bacteria in Clinical Samples. Budding AE, Hoogewerf M, Vandenbroucke-Grauls CM, Savelkoul PH.. J Clin Microbiol. 2016 Apr;54(4):934-43. doi: 10.1 128/JCM.02886-15. Epub 2016 Jan 13; IS-pro: high- throughput molecular fingerprinting of the intestinal microbiota., Budding AE, Grasman ME, Lin F, Bogaards JA, Soeltan-Kaersenhout DJ, Vandenbroucke-Grauls CM, van Bodegraven AA, Savelkoul PH.] Example 8: preferred method of treatment

The female subject is treated with a single dose of 150 mg fluconazole, followed by a daily treatment with an oral dose of 2.7 x 10 ⁹ of each of L. rhamnosus GR-1 and L. reuteri RC-14. We observed that the relative amount of vaginal L. iners increased after 28 days of treatment. Women who underwent an ART procedure (IVF or ICSI) with such increased amounts of vaginal L. Iners appeared to have an increased chance on a successful pregnancy.

Table 1 : Microbial composition of vaginal flora; prediction of chance of failure of ART procedure.

Table 4: Microbial composition of vaginal flora; prediction of chance of failure of ART procedure.

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