{"paper_id":"b3066dbb-2da8-4369-ad24-56331b5b5c44","body_text":"Infertility is multifactorial in its origin and is affected by\ndifferent factors including lifestyle, eating habits or nutrition.\nNumerous studies have shown that reduce exposure to sunlight\nand poor eating habits have led to Vitamin D insufficiency\nand/or Vitamin D deficiency, even in sunny countries\namong men and women of reproductive age ( 1 ) and this phenomenon\nis considered as one etiology for infertility ( 2 - 7 ).\nVitamin D is a fat-soluble vitamin and is considered as\nan essential nutrient required for our health. One of the\nmain functions of Vitamin D is to help with the absorbance\nof calcium and phosphate, and helps building bones\nand keeps them strong and healthy. It also blocks the release\nof the parathyroid hormone involved in reabsorption\nof bone tissue, which makes bones thin and brittle.\nConsidering these functions of Vitamin D, it plays a central\nrole in calcium and phosphate hemostasis and in turn\nis-needed for the normal mineralization of bone, muscle\ncontraction, nerve conduction, and general cellular function\nin all cells of the body including cell growth.\nVitamin D receptor (VDR) is a member of nuclear receptor\nfamily of transcription factors. It forms a heterodimer\nwith a retinoid-X receptor and binds to hormone\nresponse elements on DNA to regulate expression of specific\ngene products. At post transcriptional level Vitamin\nD regulates gene expression through microRNA-directed\nmechanisms ( 7 ). VDR is present throughout reproductive\naxis including endometrial epithelial cells, granulosa, fallopian\ntube epithelial cells and cells of cumulus oophorus\nin ovaries ( 8 ). Therefore, the reproductive axis is considered\nas one of the target organs for Vitamin D ( 9 ). In this\nregard, some studies have advocated Vitamin D plays role \nin the biosynthesis of sex hormones (estrogen and progesterone) \nand also post fertilization in the process such \nas implantation ( 10 ) and production of human chorionic \ngonadotropin (hCG) ( 11 ). Considering roles of Vitamin \nD in reproductive biology, numerous studies have shown \nthe association between Vitamin D insufficiency and deficiency \nwith fertility or poor pregnancy outcomes ( 12 , \n 13 ). In this regard, Somigliana et al. ( 14 ) showed the time \nto pregnancy is longer in women with Vitamin D insufficiency. \nHowever, it is also important to note, contrary \nreports also exist in the literature ( 15 ).\nAccording to the aforementioned role of Vitamin D \nin reproduction, researchers have tried to assess the association \nbetween serum Vitamin D concentrations and \nassisted reproductive outcomes. In this regard, Pacis et \nal. ( 16 ) in their systematic review titled “Vitamin D and \nassisted reproduction: should Vitamin D be routinely \nscreened and supplemented prior to assisted reproductive \ntechniques (ART) state that Rudick et al. ( 17 ) and \nGarbedian et al. ( 18 ) have confirmed the effect of Vitamin \nD on improvement of assisted reproductive treatment outcomes, \nbut Aleyasin et al. ( 19 ) in their study showed that \nVitamin D had no significant effect on outcomes of assisted \nreproductive treatments. Pacis et al. ( 16 ) also stated \nthat in contrary to several reports stating beneficial effect \nof Vitamin D supplementation on ART outcomes “cost-\nbenefit analysis for a single ART cycle involving fresh \nsingle blastocyst embryo transfer suggests that screening \nand supplementing vitamin D prior to ART might significantly \ndecrease societal cost per ongoing pregnancy \nby implementing a simple intervention, if the magnitude \nof the observed effect was confirmed in future studies”. \nSurprisingly the study by Anifandis et al. ( 20 ) conducted \non women who were candidates for assisted reproductive \ntreatments showed that the increase in serum level of Vitamin \nD was associated with decrease in the quality of \nembryos and the rate of achieving biochemical and clinical \npregnancy. Therefore, taking into consideration the \ncontroversial results about the effect of Vitamin D supplementation \nand according to Vanni study ( 21 ) stating \nthat the effect of Vitamin D on ART outcomes is not clear \nand should be evaluated in different populations by randomized \ncontrolled trial and cohort studies, hence current \ntrial is very important and valuable.\n\nThe clinical trial study was approved by Ethical Committee \nof Isfahan University of Medical Sciences and \nwas registered in Iranian registry for the clinical trial \n(IRCT2015111124999N1) and was designed to be carried \nout at infertile couples that referred to Isfahan Fertility and \nInfertility Center from March 2016 to June 2016 and candidate \nof ICSI. Female with age ranging from 18 to 38 years \nwho had Vitamin D level below 30 ng/ml without symptom \nof Vitamin D deficiency participated in the study.\nBased on the ethical committee, initially individuals were \nquestioned regarding clinical symptom of Vitamin D deficiency \nand individuals with these symptoms were excluded \nfrom the study, as Vitamin D treatment was mandatory for \nthese individuals. Additionally, to roll out effects of male \nfactor infertility and advance maternal age, couples with \nabnormal semen parameter based on WHO (2010) and/or \nfemale age greater than 38 were also excluded from the \nstudy. Other exclusion criteria were: secondary female \ninfertility, polycystic ovarian disease, endometriosis, congenital \nor acquired uterine malformations, drugs consumption \nthat would affect metabolism and Vitamin D absorption \nsuch as Carbamazepine and Phenobarbital Phenytoin, \nbody mass index of lower 18 or higher 30 kg/m 2  and hypothyroidism. \nCouples at risk of ovarian hyper stimulation \nsyndrome or poor endometrium (less than 7 mm or grater \n14 mm) were also excluded during the course of the study, \nsince all the embryos for these case were vitrified\nInitially 159 couples were interviewed based on Vitamin \nD level below 30 ng/ml. Fifty one couples were excluded \nbased on exclusion criteria. All of the participants \nentered the study after giving written informed consent \nand were allowed to leave the study at any desired time.\nSix couples were also excluded for other reasons including \ndeclining to participate. The remaining 108 couples \nwere randomly divided into Vitamin D and placebo \ngroups based computer-generated or random allocation \nsoftware with one block ( Fig .1 ). Participants or Vitamin D \ngroup received a weekly dose of 50000 units of Vitamin D \nsupplementation or placebo for six weeks as pearls orally. \nBoxes containing Vitamin D and Placebo peals were labeled \nbased on random allocation number, except the two \nindividuals allocating the Vitamin D and placebo, participants, \nclinician and in vitro fertilization (IVF) laboratory \npersonnel were all blind to the study. Administration of \nVitamin D or placebo started on the second day of the \nlast menstrual period (LMP) prior to ICSI cycle and continued \nto day of hCG administration which was around 6 \nweeks. Vitamin D (50000 units) and placebo pearls were \npurchased from Zahravi (Tabriz, Iran).\nFlow diagram of the progress through the phases of a 2-group parallel \nrandomized trial.\nSerum Vitamin D was assessed by high-performance \nliquid chromatography and defined based on couple’s \ninformation before starting the trial and also six to eight \nweeks after treatment, on the day of ovum pick. All the \nVitamin D assessment was carried out at a single laboratory. \nThe codes were unraveled after completion of data. \nSemen parameters, including volume, sperm density, percentage \nmotility and normal morphology were also defined \nbased on WHO (2010) manual.\nOvulation induction: all the participants received a combination \nof recombinant follicle-stimulating hormone (FSH) \nand human menopausal gonadotrophins (hMG) and were \nfollowed by sequential vaginal ultrasound. Gonadotropin releasing \nhormone (GnRH) antagonist was administered when \nsize of dominant follicles was around 12-14 mm and continued \nuntil the day of hCG administration. On the day of hCG \nadministration, number of follicles greater than 12 mm and \ntype of endometrium were also defined and recorded. Type \nof endometrium was defined according to study by Zhao et \nal. ( 22 ), briefly: cycles were divided into 3 groups depending \non the thickness (group 1: =7 mm; group 2: >7 mm to =14 \nmm; group 3: >14 mm). Each group was subdivided into \nthree groups according to the endometrial pattern as follows: \npattern A (a triple-line pattern consisting to a central hyperechoic \nline surround by two hypoechoic layers); pattern B \n(an intermediate isoechogenic pattern with the same reflectivity \nas the surrounding myometrium and a poorly defined \ncentral echogenic line); and pattern C (homogenous, hyperechogenic \nendometrium). Based on exclusion criteria individuals \nwith endometrium thickness of less than 7 mm and \ngrater 14 mm were excluded from the study. Induction of \novulation was induced with administration of 10000 IU hCG \nwhen dominant follicles reached size of 17-18 mm. vaginal \nultrasound ovum pick up was performed 36 hours post hCG \nadministration. Standard ICSI program was carried out using \nG-V series (VitroLife, Guttenberg).\nNumbers of oocytes were recorded on the day of oocyte \nretrieval. All the couples underwent ICSI based Isfahan \nFertility and Infertility policy. Fertilization rate was calculated \nbased on the number of 2PN observed over the \nnumber of injected oocytes. On day 3, embryos were \nscored for the number of blastomeres, blastomere regularity \nand percentage cytoplasmic fragmentation. Embryos \nwere considered as \"good quality\" that had between 6-8 \nblastomeres with even size and less than 25% fragmentation. \nThese outcomes were taken as primary outcomes.\nß-hCG greater than 20 IU was considered as chemical \npregnancy and clinical pregnancy was defined as pregnancy \ndiagnosed by ultrasound through visualization of \none or more gestational sac. Of note, multiple gestational \nsacs were considered as one clinical pregnancy. Therefore, \nclinical pregnancy rate was defined as the number \nof clinical pregnancy per 100 embryo transfer. These outcomes \nwere considered as secondary outcomes.\nGathered data were analyzed using SPSS for Windows \n(version 16, SPSS Inc., Chicago, IL, USA). Continuous \nvariables between two groups were compared with the independent \nt test, and categorical variables were compared \nwith the chi-square test.\n\nIn the present study, the mean age of women in the \nintervention group was 31.9 ± 4.2 years and in the control \ngroup was 30.8 ± 4.4 years. The mean of body \nmass index (BMI) in the intervention group was 23.9 \n± 2.1 and in the control group was 23.8 ± 1.9 and statistical \nanalysis showed no significant difference between \nthe demographic characteristics and the BMI \nof the intervention and the control group (P>0.05, \n Table 1 ). No statistical difference was observed for \nmale age, educational and duration of infertility and \nnumber of previous ART cycles. Therefore, these data \nsuggest that the samples were randomly allocated into \nthe two groups and both groups were similar. We also \nassess semen parameters between the two groups and \nno statistical difference was observed between the \ntwo groups. Comparison of semen parameters including \nsemen volume, sperm concentration, motility and \nmorphology revealed no statistical differences between \nthe two groups (data not shown).\nComparison of basal and clinical characteristics of couples in Vitamin D and Placebo groups\nART; Assisted reproductive techniques and *; Data are presented as mean ± SD.\nThe primary serum Vitamin D levels of the intervention \nand the control group were 14.4 ± 6.6 ng/ml and \n12.7 ± 6.4 ng/ml, respectively. The differences between \nthe two groups were insignificant. Six weeks after treatment \nwith Vitamin D or placebo, the level of Vitamin D \nsignificantly raised to 37.1 ± 7.7 ng/ml in the Vitamin D \ngroup while it remained low (13.6 ± 6.6 ng/ml) in the \nplacebo group ( Fig .2 ). Unlike in the Vitamin D group, in \nthe placebo group the difference before and after 6 was \ninsignificant.\nRegarding the ICSI primary outcomes, Table 2 \nshowed that the mean number of retrieved oocytes \nin the intervention or Vitamin D group was 9.42 ± \n4.4 and in the control group was 8.72 ± 5, and their\ndifference was not statistically significant (P>0.05). \nPercentage of type A endometrium on the day of hCG \ninjection was 81% and 55.8% in Vitamin D and placebo \ngroups, respectively and the difference between \nthe two groups was statistically significant (P<0.05). \nThe rate of fertilization in the Vitamin D group was \n68.80% and in the control group was 68% and the difference \nwas not statistically significant. The rate of \ngood quality embryo on day3 in the Vitamin D group \nwas 59.9 and in the control group was 53.59% and the \ndifference was not statistically significant (P=0.36, \n Table 2 ). We also categorize the individuals based on \nvitamin D deficiency (<10 ng/ml) and insufficiency \n(10-30 ng/ml) and compared the primary outcomes in \nthe two categories and except for type endometrium, \nno difference was observed between the two groups \nfor primary outcomes.\nComparison of ICSI outcomes in Vitamin D and placebo groups\nICSI; Intracytoplasmic sperm injection.\nComparison of serum Vitamin D levels in Vitamin D and placebo \ngroups before and after intervention.\nAccording to the results, chemical pregnancy was defined \nby positive ß-hCG in the intervention and control \ngroups were 47.6 and 25.5%, respectively, and the difference \nbetween both groups was statistically significant \n(P=0.013,  Fig .3 ). The rate of clinical pregnancy \nin the intervention group was 38.1% and in the control \ngroup was 20.9% and statistical analysis revealed \na significant difference between both groups (P=0.019, \n Fig .4 ).\nComparison of chemical pregnancy rate assessed by beta-human \nchorionic gonadotrophin (ß-hCG) in Vitamin D and placebo group.\nComparison of clinical pregnancy rate in Vitamin D and placebo groups.\n\nBased on background studies Vitamin D plays an imperative \nrole in reproduction and therefore, assessment \nof Vitamin D and thereby Vitamin D supplementation is \nbecoming part of daily practice. However, role of Vitamin \nD supplementation during assisted reproductive management \nremains controversial and there appear to be more \nroom for further study and to evaluate which parameters \nare most affected by Vitamin D deficiency and thereby supplementation. \nPart of these controversies may be related to \nconfounding factors affecting both Vitamin D levels and \nassisted reproductive outcome. An example of these confounding \nfactors is the seasonal effect on Vitamin D level \n( 6 ), therefore, in this study; the effort was taken so that \nsampling, measurement of serum Vitamin D level and supplementation \ntook place during spring and early summer.\nThe results of this study showed that despite similar \ndemographic and fertility characteristics between the \ntwo groups, Vitamin D supplementation significantly improves \nserum Vitamin D level in comparison to placebo \ngroup and this observation is in line with previous studies \nin this filed ( 23 - 25 ).\nComparing the mean value of serum Vitamin D between \nboth groups before intervention revealed no significant difference between the two groups and 50000 \nunits of Vitamin D supplementation per week for 6 \nweeks based on the previous study by Diamond et al. \n( 24 ) resulted in significant increase in serum Vitamin D \nlevel compared to before treatment and also compared \nto placebo group. Indicating that the level of Vitamin \nD increased to higher than 30 ng/l, the cut of value for \nVitamin D deficiency. The outcome of the study is in \naccordance with previous report Aflatoonian et al. ( 23 ) \nand Spedding et al. ( 25 ), indicating that this dosage of \nVitamin D supplementation was effective in improving \nthe level of serum Vitamin D.\nOur results also reveal that the improved Vitamin D level \nis also associated with significant difference observed \nin type of endometrium but no difference was observed \nbetween other assessed parameters, including percentage \nof mature oocytes, fertilization rate and embryo quality. \nThese observations are in concordance with previous report \nby Asadi et al. ( 26 ) and Rudick et al. ( 17 ). In this regard, \nKinuta et al. ( 27 ) show that VDR null mice present \nuterine hypoplasia. This phenomenon has been related to \nregulation of expression P450 aromatase activity through \n CYP19  gene containing a Vitamin D element in its promoter. \nThese authors state that “the action of Vitamin D \non estrogen biosynthesis was partially explained by maintaining \ncalcium homeostasis. However, direct regulation \nof the expression of the aromatase gene should not \nbe neglected”. But, since, the endometrium in individual \nundergoing ovarian hyperstimulation is confronted with \nhigh level of estrogen in both groups, and the difference \nin endometrial quality might be due to altered calcium homeostasis \nin the uterus, but this proposition needs further \nexploration and validation. It is important to note that as \none of the shortcomings of this study, was lack of assessment \nof estrogen level, but it is also important to consider \nthat we, like others ( 28 ) did not observe any difference \nin the number of follicle and number of oocyte retrieved \nbetween the two groups.\nAssessment of ICSI outcome in accordance with literature \nshowed that improved Vitamin D has no effect on fertilization \nand embryo quality on day 3. In contrary to our \nresults and similar studies in this filed, only one study suggest \nthat high concentration Vitamin D reduces embryo \nquality score following ICSI ( 20 ). These authors suggest \nthat glucose provides an essential substrate for cumulus-\noocyte complex (COC) and propose that Vitamin D may \nhave a physiological effect on insulin and glucose metabolism \nin a manner that remains to be elucidated. They \nbelieve increase follicular Vitamin D level decreases the \navailability of glucose to the COC and they state that this \nproposition may account for negatively correlation with \nembryo quality and FF Vitamin D levels which opposes \nour findings and findings of Polyzos et al. ( 29 ), Ozkan et \nal. ( 28 ), and Rudick et al. ( 17 ) that believe the deleterious \neffect of Vitamin D deficiency is mediated via on endometrial \nreceptivity rather than reduced embryo quality \ndue to high Vitamin D level. It is important to note the \nbased on their figures number of individuals presenting \nlower than 15 and 40 ng/ml Vitamin D are very small.\nAnother major finding of the present study was the difference \nobserved in rates of chemical and clinical pregnancies. \nIn this study rates of chemical and clinical pregnancy \nrates relative to control group was improved by 10.7% \n(47.6 vs. 25.5) and 82% (38.1 vs. 20.9), respectively. These \nresults are in accordance with several previous studies, suggests \nthat probably Vitamin D improves ICSI in term of \nboth chemical and clinical pregnancy rates ( 28 - 30 ). Based \non the literature and transfer of embryos from donor cycle, \nit appears that improved effect is very likely related to the \nimproved quality of the endometrium, as also was observed \nin this study and by other authors ( 17 ,  26 ,  29 ).\nThese improved effect has been postulated to be related \nto mechanisms including i. Miss regulation of NK cell \nactivity, ii. Immunomodulatory role during implantation \nand recurrent miscarriage, iii. Regulation of cross talk \ninvolved between embryos and endometrium which consequently \nregulates of HOXA10 involved in embryo implantation. \nIt has been shown that endometrial HOX10A \nexpression increase in parallels that Vitamin D receptor \naround time of implantation, at the time of maximal endometrial \ndifferentiation ( 16 ,  31 ). Indeed, increase quality \nof endometrium, which is reported to be lower in Vitamin \nD deficient individuals is also related to proper differentiation \nof endometrial cells ( 17 ).\n\nResults of the present study showed that consuming Vitamin \nD supplementation could be effective in improving \nthe clinical outcome of ICSI. Based on literature this effect \nis very likely to be attributed to local effect of Vitamin \nD on endometrium.","source_license":"CC-BY-4.0","license_restricted":false}