{"paper_id":"657a1612-eb83-4c14-8218-9140cba4a17e","body_text":"Infertility is a widespread problem, which affects many\nhumans in the world. Today approximately 20% of couples are facing this problem. While more than half of them\nare seeking treatment options, approximately one-quarter\nof them accept child absenteeism ( 1 ). Generally, infertility is defined as the inability of couples to get pregnant\nafter one year of regular unprotected sexual intercourse\n( 2 ). It could be the result of a disease, stressful lifestyle,\nconsumption of unhealthy foods and chemical medicines\nand exposure to industrial and environmental pollutants\nor other reasons ( 3 ). The most effective and costeffective\nway to prevent fertility problems or to treat these issues\nis nutritional modifications. Different food supplementations have significant roles in both prevention and treatment of infertility by their impact on the female and male\nreproductive systems. For instance, a deficiency in some\nvitamins and minerals can lead to infertility, and in fact,\nthere have been recent reports suggesting a role for vitamin D in infertility ( 4 )\nThe role of vitamin D in biological processes such as\ncell growth, metabolism modification, especially insulin\nfunction, autoimmune system and cardiovascular health\nis well known ( 5 ). This vitamin plays its role by interacting with vitamin D receptors (VDR) on various organs in\nthe body ( 6 ). The presence of VDR in reproductive tissues\nsuch as testis, placenta, uterus and ovary has lead to the\npossibility that this vitamin is involved in reproductive\nprocesses as well ( 7 ). Disorders such as reduced fertility,\ndiminished mating success, increased pregnancy complications, gonadal insufficiency, hypogonadism, uterine\nhypoplasia, impaired folliculogenesis ( 8 ) and infertility caused by vitamin D deficiency have been reported in\nanimal models and human ( 9 ). There are several studies,\nwhich support the role of this vitamin in calcium transport\nthrough the placenta ( 10 ), placental steroidogenesis ( 11 )\nand decidualization of endometrium ( 12 ). In addition, its\nfunction as a regulator of key target genes, which are related to implantation and establishment of the fetoplacental unit ( 13 ) has been identified.\nIt is believed that the vitamin D level in follicular fluid\ncan be associated with its level in the body resources ( 14 ).\nThere are contradictory results regarding the impact of\nthis vitamin on the number of oocytes ( 8 ,  14 ) and embryo\nquality ( 15 ,  16 ) in assisted reproduction technique (ART).\nFollicular fluid, derived from both the follicular cell secretions and plasma ( 17 ), can be an important indicator of\nvitamin D levels, as it has been shown that serum vitamin\nD levels are related to the amount of this vitamin in the\nfollicular fluid ( 15 ).\nThe presence of follicular fluid in many species shows\nits potential role in ovarian physiology, steroidogenesis\n( 18 ), follicular growth and ovulation, oocyte maturation\n( 19 ), and their transmission to fallopian tube ( 17 ). As follicular fluid provides a suitable environment for optimal\ngrowth of oocytes, which can have a direct effect on fertility ( 20 ), this study aimed to investigate the effects of\nserum and follicular levels of vitamin D on fertility and\nART outcomes.\n\nA prospective cohort study was performed on 150 women aged 18-40 years old with primary\ninfertility, who had undergone assisted reproductive treatments [intracytoplasmic sperm\ninjection +  in vitro  fertilization (ICSI+IVF)] at Isfahan Fertility and\nInfertility Center, Isfahan, Iran, from April to September 2015. A simple sampling design\nwas used. Women who met the inclusion criteria were included in the study.\nThe inclusion criteria for this study consisted of female infertility, lack of endocrine\ndisorders such as Cushing’s syndrome, Hyper or Hypothyroidism, hyperprolactinemia ( 8 ), body\nmass index (BMI) 18-29 kg/m 2  ( 20 ), lack of congenital uterine anomalies and\nendometriosis ( 20 ), and not consuming drugs affecting vitamin D metabolism ( 21 ). The\nfollowing formula was used for calculating the sample size:\nz = 1. 96\ns = an estimate of the standard deviation of vitamin D level\nd= accuracy that considered 0.16.\nAfter receiving the standard long gonadotropin-releasing hormone (GnRH)-a protocol by all the subjects,\nBuserelin Acetate 0.5 mg/day was injected intramuscularly on day 20-21 of menstrual cycle and 0.25 mg/day\nafter mensuration until ovum pick up day. Then a subcutaneous injection of 75 IU/day recombinant folliclestimulating hormone (FSH) was administered for ovarian stimulation. When at least two follicles reached 18-2 mm, human chorionic gonadotropin (HCG) 10,000 IU\nwas administered through an intramuscular injection. After 34-36 hours, ovum was picked up. All the participants\nwere followed by sequential vaginal ultrasound.\nOn the same day of ovum pick up, follicular fluid and\nserum samples were collected to determine the level of\n25-OH vitamin D (HPLC system, euro immune kit, Gerrmany). Vitamin D levels were defined as sufficient (30-\n100 ng/ml), insufficient (10-30 ng/ml), or deficient (<10\nng/ml). Fertilization was performed in the lab by an expert embryologist and the success rate of fertilization and\nembryo quality were investigated according to the number of blastomeres and fragmentation rate. Good-quality\nembryos (7 or more blastomeres and >20% fragmentation\nrate) were transferred to uterine 3 days after fertilization.\nPregnancy was detected by serum β-hCG analysis (Electrochemiluminescence method, Roche, Germany) two\nweeks after embryo transfer, and a transvaginal ultrasound\nscan was employed at 3-4 weeks later to detect the intrauterine gestational sac. Itn this study, after ovum pick ups\nin all the women, luteal phase was supported with 400 mg\nsuppository vaginal progesterone (cyclogest) twice per\nday for 10-12 weeks after pregnancy ( Fig .1 ).\nStudy flowchart. OHSS; Ovarian hyperstimulation syndrome.\nThis study was approved by Ethics Committee of Isfahan University of Medical Sciences, Isfahan, Iran (IR.\nMUI.REC.1394.3.147). A written informed consent was\ntaken from each of the participants of this research.\nData were analyzed by SPSS software (version 16,\nSPSS Inc., Chicago, Ill., USA) and Chi-square and Spearman correlation coefficient. A value of P≤0.05 was considered statistically significant.\n\nThis study included 150 infertile women. Thirteen of them were excluded from the study due to ovarian\nhyperstimulation syndrome (OHSS) or because their\nendometrium was not ready for emberyo transfer.\nUltimately, data analysis was performed on 137\npatients. The patients’ demographic characteristics are\nsummarized in  Table 1 . The age of the females ranged\nfrom 21-40 with an average age of 30.30 ± 4.75 years.\nThe mean of vitamin D level of follicular fluid and the\nserum was 26.99 ± 24.32 ng/ml and 26.37 ± 24.36 ng/\nml, respectively. Also, there was a significant difference\nbetween the levels of vitamin D in serum and follicular\nfluid (r=0.711, P<0.001).\nThe results show that 45.8% of the subjects with positive\nbiochemical pregnancy had serum vitamin D levels above\n30 ng/ml, while 80.5% of the women with negative\npregnancy rate were in the group with vitamin D levels\nless than 10 ng/ml. The statistical analysis indicated that\nthere was a positive association between serum vitamin\nD levels and the incidence of biochemical pregnancy\n(P=0.008) and clinical pregnancy (P=0.017,  Table 2 ).\nIn addition, in 44.4% of the females who had a\npositive biochemical pregnancy, the vitamin D level in\nfollicular fluid was higher than 30 ng/ml, while in 84.6%\nof the women who had less than 10ng/ml vitamin D in\ntheir follicular fluid, a successful pregnancy did not\noccur. Statistically, a positive association was found\nbetween follicular fluid vitamin D levels and pregnancy\n(biochemical pregnancy, P=0.003, chemical pregnancy,\nP=0.001) ( Table 2 ).\nBaseline characteristics of study population (n=137)\nBMI; Body mass index.\nTable 3 shows that there is no association between\nthe serum level of vitamin D and the quality of embryo\n(r=0.126, P=0.125) or fertilization rate (r=0.019,\nP=0.082). Similarly, no associations were observed\nbetween the follicular fluid level of vitamin D and embryo\nquality (r=0.133, P=0.106) or fertilization rate (r=0.154,\nP=0.059).\nSerum and follicular fluid vitamin D levels and biochemical and clinical pregnancy rate\nData are presented as n (%). *; P≤0.05 was considered significant.\nFollicular fluid vitamin D levels and embryo quality and fertility rate\nData are presented as mean ± SD. *; P≤0.05 was considered significant.\n\nIn this study, we sought to elucidate one of the most\ncontroversial issues in fertility, which is whether vitamin\nD affects assisted reproduction outcomes. Over the\npast decades there has been extensive investigation\non the physiological roles of 25-OH vitamin D on\nART outcomes, but the results of previous studies\nare very heterogeneous ( 22 ). This heretogenousity\ncan be due to various factors affecting vitamine D\nlevels including diet and the degree of exposure with\nsunlight ( 23 ). Nonetheless, independently of these\nfactors, animal experimental studies have shown that\nvitamin D deficiency may affect fertility through Ca\ndependent/independent hemostasis ( 24 ). However, most\nreproductive consequnences of viatmin D deficiency are\ncorrected. Consequently, this prospective cohort study\nwas performed to evaluate the association of vitamin D\nlevels in follicular fluid and serum on both biochemical\nand clinical pregnancy outcomes. We also evaluated the\nassociation between these two parameters with embryo\nquality and fertilization rates among participants.\nIt is noteworthy that the relationship between the level\nof 25-OH vitamin D in follicular fluid is a reflective of\nstores of vitamin D in the body ( 8 ,  14 ). Moreover, serum\nand follicular fluid 25(OH)D are directly related to each\nother ( 25 ).\nOur results showed a significant association between the\nserum and follicular fluid vitamin D levels and pregnancy\nrate, which is in agreement with several previous published\nwork ( 4 ,  8 ,  16 ,  26 ,  27 ), however, it is in contrast to other\nstudies, which have reported no association ( 14 ,  25 ,  28 -\n 30 ) or inverse relation ( 15 ).\nWe also concluded that Vitamin D status is not associate\nwith embryo quality and fertilization rates, despite the\nfact that association ofthe fertilization rate was close to be\nsignificant. These results are in accordance with Rudick\net al. ( 16 ) and Aleyasin et al. ( 31 ). While Anifandis et al.\n( 15 ) found that higher levels of this vitamin have a negative\nimpact on embryo quality and therefore on IVF outcome.\nRudick et al. ( 16 ) showed an association between\nvitamin D and IVF success rate among non-Hispanic\nwhites but not in Asians. They concluded that there was a\nstatistically significant impact of race on the relationship\nbetween these two parameters. Our current finding are\nin contrast with previous results of investigations in Iran\n( 14 ,  29 ) that suggested no relationship between vitamin\nD levels and the outcomes of ART. Definitely, the most\nsurprising results were observed by the Anifandis et al.\n( 15 ), as they reported an excess level of vitamin D in\ncombination with a decreased level of follicular fluid\nglucose have an adverse effect on ART outcomes of\ninfertile Greek women.\nConsidering the substantial discrepancies with published works, these results add to the\nliterature the potential role of vitamin D on pregnancy rate among infertile couples\nundergoing infertility treatments. The possible mechanism can be explained as follows:\nfirstly, vitamin D has been diagnosed as a factor, which affects endometrium receptivity.\n1,25-dihydroxy vitamin D 3  (1,25[OH] 2  D 3 ) is produced in\nendometriotic cells in response to interleukin B1, which is secreted by blastocyst. This\nenzyme binds to VDRs on the endometrium and regulates the expression of genes involved in\nimplantation and placental development ( 32 ). Additionally, vitamin D plays a critical role\nin upregulation of transcription of  HOXA10  gene, an important gene\nparticipating in both placentation and implantation ( 33 ). It is important to point out that\n HOXA10  gene can be activated by interaction with vitamin D ( 34 ).\nSecondly, the influence of vitamin D on development of follicles and embryo has been\npreviously reported ( 23 ). This vitamin also stimulates the production of estradiol, estrone,\nand progesterone and the enzymes that are responsible for the production of these hormones\nhave vitamin D response element in their promotors ( 24 ). Additionally, anti mullerian\nhormone (AMH), a marker of ovarian reserve, has an inhibitory effect on the primordial\nfollicle recruitment during folliculogenesis. It has been shown that there is a functional\nVDR element (VDRE) in the promoter of  AMH  gene ( 24 ) and that AMH is\npositively affected by vitamin D. Therefore, defects in VDR or its deficiency in the body\ncan retard follicle development and oocyte maturation ( 14 ). Thus, it is not surprising to\nsee some reports regarding the relationship between level of vitamine D and low ovarian\nresponse, as well as the fact that viatmine D supplementation increases AMH level ( 24 ).\nFinally, vitamin D plays a vital role in gestation\nand maintaining a healthy pregnancy. The association\nbetween a decreased level of vitamin D and a higher\nrisk of gestational diabetes and preeclampsia has been\ninvestigated by several studies ( 35 ).\nConsidering all the above mentioned findings, it is not\nunexpected to observe that vitamin D plays a crucial role\nin fertility outcomes. Therefore, the discrepancy within\npublished works can be explained by other confounding\nfactors, such as a source of vitamin D (diet, exposure\nto the sun, former supplementation), lifestyle, ethnicity,\nage, BMI, seasonal effect, and involvement of other\novarian factors responsible for this procedure.\nIn this research certain limitations should be considered;\nalthough we investigated maternal vitamin D status, the\npaternal vitamin D concentration needs to be assessed\nsimultaneously. There are numerous studies showing that\ndeficiency in vitamin D not only effects sperm parameters\nbut also affects sperm DNA integrity, which subsequently\nwill affect embryo developmental competency\n( 35 ). Additionally, there is a lack of monitoring and\nmeasurement of vitamin D levels during pregnancy until\ndelivery. We analyzed our data in terms of biochemical\nand clinical pregnancy to help better understand these\nissues. And finally, although we provided sufficient results\nthrough this study, it is nearly impossible to measure all\nthe various confounding factors.\nIndeed, a possible association has been reported among\nvitamin D and small for gestational age (SGA) infants ( 37 -\n 39 ), preeclampsia ( 35 ), and gestational diabetes mellitus\n(GDM) ( 40 ). Therefore, the side effect of vitamin D\nsupplementation during pregnancy should be considered\nbefore suggesting its widespread consumption.\n\nThe findings of this study revealed that there is a positive\nassociation between serum and follicular fluid vitamin D\nlevels and the success rate of biochemical and clinical\npregnancy. However, there was no significant relationship\nbetween vitamin D level of follicular fluid and embryo\nquality or fertilization rate. Vitamin D supplementation is\nsuggested to increase the level of this vitamin to a normal\nrange in women with an insufficient level of vitamin D for\nachieving successful biochemical and clinical pregnancy.","source_license":"CC-BY-4.0","license_restricted":false}