Clinical Tests to Predict the Success of Assisted Reproductive Techniques
Condition: Infertility
MeSH terms: Infertility
Study type: Observational

Summary: 
      Today, it is evident that vitamin D has more widespread effects than the classical actions
      related to bone mineralization and calcium homeostasis. Vitamin D deficiency results in
      impaired reproductive performance in various species of animals, and recently the
      investigators have shown that the Vitamin D receptor (VDR), activating (CYP2R1, CYP27A1,
      CYP27B1) and inactivating (CYP24A1) enzymes are expressed in the human testis, epididymis,
      seminal vesicle, prostate and spermatozoa. Functional studies showed that activated vitamin D
      increases intracellular calcium and sperm motility in mature spermatozoa, and hence may be
      important not only for spermatogenesis but also for sperm function. Any test that might
      assist in guiding the treatment of the infertile couple would be beneficial both for most
      infertile couples and the society in general. The fact that vitamin D may play a role for
      human semen quality are now being tested clinically. If vitamin D supplementation proves
      efficient this opens for the first time for a causal, safe and cheap treatment of at least
      some cases of "idiopathic" impaired semen quality. This may also have consequences in the in
      vitro setting as activated vitamin D may be used to select high quality sperm during assisted
      reproductive techniques. The presence of the vitamin D receptor and vitamin D metabolizing
      enzyme CYP24A1 in particular is able to discriminate spermatozoa from normal and infertile
      men. CYP24A1 is expressed at the annulus of normal sperm, but it is virtually absent from
      spermatozoa from infertile men. This indicates that CYP24A1 expression may assist in
      predicting the chance of success by using insemination (IUI), IVF or ICSI. CYP24A1 expression
      is induced by activated vitamin D, which indicates that other VDR activated genes also may
      serve as positive predictive markers of fertility. In addition, vitamin D metabolites and
      other factors in the female reproductive tract will be measured to determine if they alone or
      in combination with other markers can determine whether the best solution for the infertile
      couple would be to do IUI, IVF, or ICSI. The suggested clinical trial may therefore be able
      to evaluate several secondary endpoints in addition to CYP24A1 in our search for predictive
      markers for fertilization. For instance several biomarkers in serum, seminal plasma or
      follicular fluid in conjunction with genetic polymorphisms in several genes important for
      reproductive function.
    

Detailed description: 
      Background Today, it is evident that vitamin D has more widespread effects than the classical
      actions related to bone mineralization and calcium homeostasis. Vitamin D deficiency results
      in impaired reproductive performance in various species of animals, and recently the
      investigators have shown that the vitamin D receptor (VDR), activating (CYP2R1, CYP27A1,
      CYP27B1) and inactivating (CYP24A1) enzymes are expressed in the human testis, epididymis,
      seminal vesicle, prostate and spermatozoa. Functional studies showed that activated vitamin D
      increases intracellular calcium and sperm motility in mature spermatozoa, and hence may be
      important not only for spermatogenesis but also for sperm function. A cross sectional study
      of 300 young healthy Danish men showed that men with lower levels of serum 25-hydroxyvitamin
      D have significantly lower number of normally developed and motile spermatozoa. Hitherto,
      most cases of male infertility have been classified as "idiopathic", and infertile couples
      have been referred to symptomatic treatment at infertility clinics. These fertility
      treatments are often physically demanding for the female partner as well as expensive for the
      health care system. Any test that might assist in guiding the treatment of the infertile
      couple would be beneficial both for the infertile couples and the society in general. Our
      findings that vitamin D may play a role for human semen quality are now being tested
      clinically. If vitamin D supplementation proves efficient this opens for the first time for a
      causal, safe and cheap treatment of at least some cases of "idiopathic" impaired semen
      quality. This may have consequences as vitamin D may be used to select high quality sperm
      during assisted reproductive techniques. The presence of the vitamin D receptor and vitamin D
      metabolizing enzymes in a particular CYP24A1 is able to discriminate sperm from normal and
      infertile men. CYP24A1 is expressed at the annulus of normal sperm but is virtually absent
      from spermatozoa from infertile men. This indicates that CYP24A1 may be used as a marker to
      discriminate between good and unhealthy sperm and may therefore serve as a predictive
      clinical marker for fertility. This indicates that CYP24A1 expression also may assist in
      predicting the chance of success by using insemination (IUI), IVF or ICSI. CYP24A1 expression
      is induced by activated vitamin D through the VDR, which indicates that other VDR activated
      genes also may serve as positive predictive markers of fertility.

      In addition to the simple use of VDR, CYP24A1 and other vitamin D regulated genes in human
      sperm as clinical markers, it is also important to determine the function of vitamin D in
      reproduction. In order to understand this, it is important to determine the concentration of
      vitamin D metabolites in the male and female reproductive tract. For instance if vitamin D
      metabolites are undetectable in the male reproductive tract but measurable in the female
      reproductive tract then it may be important for signaling to the capacitated (activated)
      spermatozoa. The different vitamin D metabolites and other factors in the female reproductive
      tract will be measured to determine if they alone or in combinaton with other markers may be
      good predictors of the success following IUI, IVF, or ICSI treatment. The suggested clinical
      trial may therefore be able to evaluate several secondary endpoints in addition to CYP24A1 in
      our search for predictive markers for fertilization. For instance several biomarkers in
      serum, seminal plasma or follicular fluid in conjunction with known polymorphisms in several
      genes important for reproductive function. For instance genetic variation in FSH signaling.
      Single nucleotide polymorphisms (SNPs) related to genes encoding the FSHβ subunit (FSHB) and
      the FSH receptor (FSHR) affect FSH production (FSHB c.-211 G>T) and sensitivity/expression of
      its receptor in vitro (FSHR c.2039A>G & FSHR c.-29G>A). FSHR c.2039A>G, but not FSHR
      c.-29G>A, is associated with increased FSH levels in adult women, while there are conflicting
      results on FSHB c.-211 G>T (7;8). May these polymorphisms and other specific polymorphisms
      affect male and female fertility potential, semen quality and reproductive hormones.

      SETTING, SCIENTIFIC PLAN AND RECRUITMENT Participants will be included among infertile
      couples referred for IUI, IVF or ICSI at Dansk fertilitetsklinik. The design is a
      prospective, blinded, single center cohort study. The investigation of all samples will be
      blinded since investigators have no information about the clinical data, treatment
      failure/succes. all participants will be followed until 9 months after their treatment to
      evaluate live birth rate, abortions etc.

      PARTICIPANTS All referred infertile couples will be invited to participate, however both
      partners should be > 18 years and the women < 43 years old. women using donorinsemination
      will also be included. Anticipated 800-1000 IUI and 400 IVF/ICSI will be conducted at the
      clinic in the study period. The investigators assume that up to 600 IUI and 200 IVF/ICSI wish
      to participate. Thus, 800 treatments is the target for inclusion in the study. The
      investigators expect a small retraction rate (< 20) because of high motivation and no adverse
      effects.

      ANALYSIS Reproductive hormones, genetic analyses, endocrine disrupters and growth factors
      will be analyzed at dept. of GR,Rigshospitalet and calcium regulators including vitamin D
      metabolites at Holbæk hospital.

      SAMPLE SIZE CALCULATION AND STATISTICS The power estimate is based on the published data on
      CYP24A1 as a positive marker of semen quality. The association between clinical pregnancy and
      CYP24A1 is estimated to be comparable with the association with sperm motility. This imply
      that N=600 IUI will be enough to evaluate the effect of CYP24A1 as a marker of pregnancy and
      live birth rate because the investigators estimate that at least 12% will have a positive
      pregnancy test. When the inital collection of raw semen/percoll separated sperm and data have
      been completed an additional amount of clinical observations will be obtained after 9 months
      on live birth rate, abortions etc. Secondary analyses on the putative association between
      clinical endpoints and the expression of VDR and other Vitamin D regulated genesin sperm will
      be investigated on a randomly selected subset of samples N=300. All the listed genetic
      analyses will be conducted on all men and all the women with DNA. Follicular fluid will be
      collected during IVF or ICSI when there is no contamination with blood. Analyses of different
      markers in the follicular fluid and cells will be associated with oocyte quality, pregnancy
      and live birth rate.

      ETHICS AND SIDE EFFECTS All the patients will have finished their inital visit and
      investigations, before they are invited to participate in the study. The loss of spermatozoa
      for each man as a result of making the cytospin will not influence the successrate of IUI,
      IVF or ICSI because the investigators will take less than 2.5% of the sperm pool.

      PUBLICATION OF RESULTS All results, positive or negative will be submitted to peer reviewed
      scientific journals. Data will successively be obtained and transferred to a statistical
      database.

      Predefined subgroup analyses Female age, sperm concentration, TTP, BMI, expression of
      CYP24A1/VDR in sperm, raw semen verus percoll separated, serum vitamin D levels and the
      listed genetic polymorphisms under secondary endpoints.
    

Observational model: Cohort
Time perspective: Prospective
Sampling method: Probability Sample
Study population: 
        All infertile couples referred to Dansk Fertilitetsklinik will be invited to participate.
        Anticipated 800-1000 IUI and 400 IVF/ICSI will be conducted during the planned study
        period. The investigators assume that up to 600 IUI and 200 IVF/ICSI wish to participate.
        Thus 600-800 treatments is the estimated target for inclusion in this study. The
        investigators expect a small retraction rate (< 20) because of high motivation and no
        adverse effects.
      
Biospecimen description: 
      Not number of subject but number of cycles/treatments Serum Seminal plasma follicular fluid
      DNA granulosa cells
    
Keywords: vitamin d; fertility; markers; endocrine disruption; reproduction