{"paper_id":"c431ee6a-ccd9-4359-bf0b-fd7ed4fe9507","body_text":"Around the world, 6 to 12 percent of women of reproductive age suffer from polycystic\novarian syndrome (PCOS), a prevalent female endocrine disorder ( Bozdag  et al.,  2016 ). Some of\nits symptoms include menstrual irregularity, anovulatory infertility,\nhyperandrogenism, obesity, and metabolic disorders ( Rotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop group, 2004 ),_all\nof which may significantly affect a patient’s health and quality of life. However,\ndue to the complexity of its pathophysiology, the disease’s cause remains unclear,\nand no effective treatment has been found as of yet ( Rotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop group, 2004 ).\nStudies have shown that the endometrium may be significantly impacted by endocrine\nand metabolic irregularities in PCOS, which may lead to infertility, endometrial\ninvolvement, and issues such as impaired implantation processes ( Donaghay & Lessey, 2007 ;  Qiao  et al ., 2008 ;  Govahi  et al ., 2023 ). Impaired\nuterine receptivity is one of the leading causes of spontaneous abortion in PCOS\npatients ( Lopes  et al .,\n2011 ).\nAccording to studies, women with PCOS experience a decrease in HOXA-10, LIF, and\nβ-integrin proteins during the secretory phase ( Bergeron  et al ., 1988 ;  Apparao  et al ., 2002 ;  Cermik  et al ., 2003 ;  Daftary  et al ., 2007 ). Integrins, or CAMs, are\nbelieved to be reliable indicators of endometrial receptivity and are essential in\ncell interaction. Integrins exhibit a sandwich-like structure in the embryonic\njunction ( Wang & Armant, 2002 ;  Achache & Revel, 2006 ;  Jiang & Li, 2022 ; Zhang  et al ., 2022;\n Bajpai  et al ., 2023 ).\nAdditionally, expression of LIF mRNA in the endometrium of healthy women occurs\nduring the middle and late secretory phases of the menstrual cycle, as well as in\nearly pregnancy ( Aghajanova, 2004 ;  Kara  et al ., 2016 ;  Ajdary  et al ., 2020 ;  Fukui  et al ., 2022 ;  Bajpai  et al ., 2023 ). The\nHOXA-10 gene also has a physiological role in implantation by regulating the\nresponse of endometrial stromal cells to progesterone ( Celik  et al ., 2015 ;  Kara  et al ., 2016 ;  2019 ; Zhang  et al. , 2022;  Bajpai  et al ., 2023 ).\nDespite the ability to select high-quality embryos during assisted reproductive\nprocedures, implantation rates have remained low and have not significantly\nincreased in recent years ( Andersen  et\nal ., 2005 ;  Amjadi  et\nal ., 2019 ;  Ajdary  et\nal ., 2020 ). Uterine receptivity is a critical factor in\ndetermining the success of pregnancy and may be adjusted to increase the\neffectiveness of assisted reproductive technologies, particularly in gynecological\nconditions such as PCOS ( Donaghay & Lessey,\n2007 ). Thus, there is an increasing need for creative interventions to\ntreat this condition effectively.\nSeveral recent studies have described the relationship between vitamin D3 (VD3)\ndeficiency and the progression of insulin resistance, hypertension, dyslipidemia,\nglucose intolerance, diabetes, obesity, and metabolic syndrome ( Bahadur  et al ., 2022 ;  Morgante  et al ., 2022 ;  Simpson  et al ., 2022 ). Some\nstudies have also highlighted the higher prevalence of VD3 deficiency in patients\nwith PCOS and the relationship between this deficiency and the development of\ninsulin resistance, and metabolic and endocrine conditions in these patients ( Hahn  et al ., 2006 ;  Kotsa  et al ., 2009 ;  Wild  et al ., 2011 ;  Thomson  et al ., 2012 ;  Mousa  et al ., 2015 ;  Trummer  et al ., 2018 ). This\ntheory is supported by the idea that the VD3 receptor gene regulates approximately\n3% of the genes in the human body, including those involved in glucose and lipid\nmetabolism and blood pressure control ( Thys-Jacobs\n et al ., 1999 ). Additionally, VD3 adjusts the\nexpression of 229 genes in over 30 different tissues, such as the pancreas, liver,\nimmune cells, and ovaries ( Gatti  et\nal ., 2016 ).\nVD3 has low bioavailability due to its poor dissolution in water. Recent studies have\nshown that slow-release formulations (SRDDSs) in the drug delivery system have a\nfavorable result in slow-release speed, reducing the number of daily doses, and\nimproving bioavailability. Previous studies have shown that phospholipids can be\nused to increase the release of drugs with poor dissolution in water. In addition,\nphospholipids protect against drug degradation in the gastrointestinal tract ( Haddadzadegan  et al ., 2022 ).\nIn the PhytoSolve technique, phospholipids diffused in a very concentrated aqueous\nsolution of carbohydrate or polyol can dissolve large amounts of lipids, steroids,\nterpenes, and polar lipids ( Keyhanfar  et\nal ., 2014 ). In our previous study, we used VD3 loaded in the\nPhytoSolve formulation to treat patients with PCOS. In this study, we investigated\nthe effect of this VD3 formulation on the expression of genes involved in\nimplantation in patients with PCOS.\n\nAntibodies were obtained from Abcam (ab138002, ab153904, ab179471 Cambridge,\nUnited Kingdom) and TRIzol and Glycerol were prepared from Sigma Aldrich (St.\nLouis, MI, USA). Deionized water and SYBR Premix Ex Taq were obtained from a\nMilli-Q water purification system (Millipore, Burlington, MA, USA) and Applied\nBiosystems (Foster City, CA, USA) respectively. cDNA synthesis kits were\nprepared by Thermo Fisher Scientific (Waltham, MA, USA).\nThe preparation of PhytoSolve containing VD3 was carried out according to our\nprevious study ( Hakimpour  et\nal ., 2022 ). Briefly, 20 mg of VD3 in 2 g of medium-chain\ntriglyceride (MCT) oil, and 0.5g of phospholipid (Lipoid S75) were homogenized\nin 7.5g of polyol phase at room temperature with an Ultra-Turrax homogenizer\n(IKA T10B, Germany). The oil phase (MCT+VD3) was slowly added to the\nphospholipid and polyol phase and mixed. The final mixture was sonicated with a\nprobe sonicator (Hielscher, Germany) at 70% intensity for 10 minutes to reduce\nthe emulsion particle size. The PhytoSolve formulation was prepared using polyol\nphase, and the physicochemical properties of the PhytoSolve containing VD3 were\ndescribed in our previous study ( Hakimpour\n et al ., 2022 ).\nThirty-six 25-day-old female NMRI laboratory mice were divided into six groups:\ncontrol group, which received normal food and water without any treatment; sham\ngroup, which received sesame oil i.p for 20 days via gavage; PCOS group, which\nconsisted of animals with polycystic ovary disease without any treatment; VD3\ngroup, which consisted of animals with PCOS given VD3; PhytoSolve group, which\nconsisted of animals with PCOS given the PhytoSolve formulation without VD3; and\nthe PhytoSolve containing VD3 group, which consisted of animals with PCOS given\nPhytoSolve containing VD3. The treatment of the PCOS model groups was\nadministered via gavage starting 13 days after model induction for one week.\nInduction of PCOS in mice was performed according to the protocol described in\nour recent study ( Hakimpour  et\nal ., 2022 ). Briefly, a subcutaneous injection of a\nmixture of dehydroepiandrosterone (DHEA), sesame oil, and 95% ethanol was\nadministered for 20 days.\nAll animals were kept under standard light and dark conditions, and had free\naccess to food and water. During the treatment period, the animals’ weight was\nmeasured every two days and vaginal smears were taken daily from 10 days after\nthe first injection until the end of the experiment. All mice remained in the\nestrous cycle, and the control group was blooded on the day of estrus. Ovarian\ntissue was then separated and placed in 10% formalin. Additionally, blood was\ntaken from the animals’ hearts to check fasting testosterone and insulin levels.\nThe entire female mice were coupled with NMRI male mice. The vaginal plugs were\nexamined in the morning after finishing the treatment, and if a plug was formed,\nit was considered the first day of pregnancy. Pregnant animals were euthanized\nwith ether on day 4.5. Endometrial samples were then collected and stored at\n-80°C for RNA extraction.\nReagent TRIzol (Sigma-Aldrich) was used to extract total RNA from endometrial\nsamples based on manufacturer instructions. Technical triplicates were made for\nevery sample. In order to remove genomic DNA contamination, the entire samples\nwere incubated with DNase I (Fermentas, St. Leon- Rot, Germany). Then, for\ndetermining RNA concentration, yield, and purity, using the A260/A280 ratio\nmethod, samples were analyzed on a spectrophotometer. Using oligo dT primers\n(Metabion, Martinsried, Germany) and the SuperScript First-Strand Synthesis\nSystem (200 U/ ml, Invitrogen), total RNA was reverse transcribed. One converse\ntranscription control carried out under the aforementioned conditions without\nSuperScript II enzyme was utilized in every PCR cycle. In primary tests, using a\nconventional PCR protocol (Invitrogen), the primer pairs were tested, and the\nproducts run on agarose gel were limited to a single band of the expected size.\n Table 1  presents the gene-specific\nprimers used. Negative controls without cDNA were included in all experiments.\nqPCR reactions were performed as  previously described  ( Ajdary  et al.,  2020 ). To\nensure the removal of contaminants or primer dimers, PCR reactions’ melting\ncurves were monitored. Using the cDNA’s logarithmic dilution series, standard\ncurves were obtained for every gene. Normalization of the threshold cycle values\nwas based on the threshold value of human GAPDH. Using the comparative CT\nmethod, the data of qPCR were analyzed ( Ajdary\n et al.,  2021 ;  Govahi  et al.,  2023 ).\nThe sequence of the primers used in this study.\nIHC staining was performed to determine  LIF, HOXA10,\nβ-integrin  proteins. IHC DAB staining was used to\nobserve  LIF, HOXA10, and β-integrin  protein\nexpression. The mice underwent transcardiac perfusion with 4% paraformaldehyde\nin phosphate buffer and then slaughtered. The uterus of each mouse was removed\nand postfixed overnight. Then, they were dehydrated in an ascending alcohol\nseries, rinsed with xylene, and embedded in paraffin. Then the blocks were\ndivided into 5 µm sections. To prepare the slides for immunostaining\nanalysis, the antigen retrieval process involved soaking them in a citrate\nbuffer for 10 minutes. Triton X-100 0.3% (30 minutes) and normal goat serum (1\nhour) were used to permeabilize and block the slides, respectively. The sections\nwere incubated overnight at 4°C with primary antibodies for\n HOXA10  (rabbit an ti-HOXA10  (1:100, Abcam,\nCambridge, United Kingdom)),  LIF  (rabbit\nanti- LIF  (1:100, Abcam, Cambridge, United Kingdom)) and\n β-Integrin  (rabbit anti-\n β-Integrin  (1:100, Abcam, Cambridge, United\nKingdom)). The slices were then incubated in secondary antibody at 37°C for 90\nmin. Peroxidase-conjugated secondary antibodies were used for chromogenic\ndetection by oxidizing 3,3′-Diaminobenzidin (1:200, ab205718) according to\nmanufacturer instructions.  Hematoxylin  was used to stain nuclei\nin IHC.\nThe results were explained using mean and standard error. Statistical analysis\nwas performed using one-way ANOVA and the internal Tukey’s multiple comparisons\ntest as post hoc. A  p -value of 0.05 or less was considered\nsignificant.\nAll studies were executed in accordance with the Guide for the Care and Use of\nLaboratory Animals (National Institute of Health Publication No. 80-23, revised\n1996) and were approved by the Research and Ethics Committee at Iran University\nof Medical Sciences (IR.IUMS.REC. 1399.1069), Tehran, Iran.\n\nAssessment of the physicochemical properties of PhytoSolve containing VD3\nThe average particle size in the PhytoSolve formulation was 71.10±1.100 nm,\nand after 120 hours, 91.5% of the vitamin D3 had been released from it ( Hakimpour  et al.,  2022 ).\nPCOS model confirmation was done based on the method presented in our previous\nstudy and based on body weight, number of follicles, and insulin and\ntestosterone levels ( Hakimpour  et\nal.,  2022 ).\nAccording to  Figure 1 , the group with PCOS\nhad lower levels of  HOXA10, β-integrin,  and\n LIF  gene expression than the other groups, and this\ndifference from the control group was significant ( p <0.001,\n p <0.0001). Also, the  HOXA10,\nβ-integrin,  and  LIF  gene expression in the\nVD3 and VD3-containing PhytoSolve groups increased significantly in comparison\nto the group with PCOS ( p <0.05,  p <0.01,\n p <0.001). In addition to the expression of the\n HOXA10, β-integrin  and  LIF  genes in\nthe VD3-containing PhytoSolve group were significantly increased in comparison\nto the VD3 group ( p <0.05,  p <0.01).\nFigure 1 Gene expression  HOXA10  (A),\n β-Integrin  (B),  LIF  (C)\nin endometrium from PCOS mice. The mRNA assay were performed by\nqRT-PCR. Data was presented as Mean ± SD (n=6 per every\ngroup). Internal control was done by β-actin.\n* p <0.05, ** p <0.01,\n*** p <0.001, and\n**** p <0.0001.\nGene expression  HOXA10  (A),\n β-Integrin  (B),  LIF  (C)\nin endometrium from PCOS mice. The mRNA assay were performed by\nqRT-PCR. Data was presented as Mean ± SD (n=6 per every\ngroup). Internal control was done by β-actin.\n* p <0.05, ** p <0.01,\n*** p <0.001, and\n**** p <0.0001.\nAccording to  Figure 2 , the level of\n HOXA10  protein expression in the group with PCOS decreased\nsignificantly when compared to the control group ( p <0.0001).\nThe increase in  HOXA10  protein expression between the group\nreceiving VD3-containing PhytoSolve and the group with PCOS was also significant\n( p <0.0001). The increase in  HOXA10 \nprotein expression between the group receiving VD3 and the group with PCOS was\nalso significant ( p <0.001). The increase in\n HOXA10  protein expression in the group receiving\nVD3-containing PhytoSolve increased compared to the vitamin D3 group, although\nnot significantly.\nFigure 2 The expression of HOXA10 protein in endometrium from PCOS mice; The\nassay was performed by Immunohistochemistry technique. Data was\npresented as Mean ± SD (n=6 per every group). Internal\ncontrol was done by β-actin. *** p <0.001,\nand **** p <0.0001. A: Control, B: Sham, C: PCOS,\nD: Vitamin D, E: Phytosolve, F: Phytosolve/ Vitamin D.\nThe expression of HOXA10 protein in endometrium from PCOS mice; The\nassay was performed by Immunohistochemistry technique. Data was\npresented as Mean ± SD (n=6 per every group). Internal\ncontrol was done by β-actin. *** p <0.001,\nand **** p <0.0001. A: Control, B: Sham, C: PCOS,\nD: Vitamin D, E: Phytosolve, F: Phytosolve/ Vitamin D.\nFigure 3  shows that the level of\n β-integrin  protein expression in the group with PCOS\ndecreased significantly compared to the control group\n( p <0.0001). The increase in\n β-integrin  protein expression between the group\nreceiving VD3-containing PhytoSolve and the group with PCOS was also significant\n( p <0.01). The increase in\n β-integrin  protein expression between the group\nreceiving VD3 and the group with PCOS was also significant\n( p <0.05). The increase in  β-integrin \nprotein expression in the group receiving VD3-containing PhytoSolve increased\nagainst the VD3 group, although not significantly.\nFigure 3 The expression of β-Integrin protein in endometrium from PCOS\nmice; The assay was performed by Immunohistochemistry technique.\nData was presented as Mean ± SD (n=6 per every group).\nInternal control was done by β-actin. *p<0.05,\n**p<0.01, ***p<0.001 and ****p<0.0001. A: Control, B: Sham,\nC: PCOS, D: Vitamin D, E: Phytosolve, F: Phytosolve/ Vitamin D.\nThe expression of β-Integrin protein in endometrium from PCOS\nmice; The assay was performed by Immunohistochemistry technique.\nData was presented as Mean ± SD (n=6 per every group).\nInternal control was done by β-actin. *p<0.05,\n**p<0.01, ***p<0.001 and ****p<0.0001. A: Control, B: Sham,\nC: PCOS, D: Vitamin D, E: Phytosolve, F: Phytosolve/ Vitamin D.\nFigure 4  shows that the expression level of\n LIF  protein in the group with PCOS decreased significantly\ncompared to controls ( p <0.0001). The increase in\n LIF  protein expression between the group receiving\nVD3-containing PhytoSolve and the group with PCOS was also significant\n( p <0.0001). The increase in  LIF  protein\nexpression between the group receiving VD3 and the group with PCOS was also\nsignificant ( p <0.01). There was a significant increase in\n LIF  protein expression in the group receiving\nVD3-containing PhytoSolve against the VD3 group\n( p <0.05).\nFigure 4 The expression of  LIF  protein in endometrium from\nPCOS mice; The assay was performed by Immunohistochemistry\ntechnique. Data was presented as Mean ± SD (n=6 per every\ngroup). Internal control was done by β-actin.\n* p <0.05, ** p <0.01, and\n**** p <0.0001. A: Control, B: Sham, C: PCOS,\nD: Vitamin D, E: Phytosolve, F: Phytosolve/ Vitamin D.\nThe expression of  LIF  protein in endometrium from\nPCOS mice; The assay was performed by Immunohistochemistry\ntechnique. Data was presented as Mean ± SD (n=6 per every\ngroup). Internal control was done by β-actin.\n* p <0.05, ** p <0.01, and\n**** p <0.0001. A: Control, B: Sham, C: PCOS,\nD: Vitamin D, E: Phytosolve, F: Phytosolve/ Vitamin D.\n\nThe results of this study showed that the gene expression of  LIF,\nβ-integrin,  and  HOXA10  in the endometrial\ntissue of mice with PCOS in the PhytoSolve containing VD3 group significantly\nincreased compared to the VD3 group. Also, the expression of proteins\n β-integrin  and  HOXA10  increased in the\nPhytoSolve containing vitamin D3 group against the vitamin D3 group, although not\nsignificantly. The increase of  LIF  protein in the\nPhytoSolve-containing vitamin D3 group was significant against the vitamin D3\ngroup.\nStudies have reported that the formulation of drugs based on lipids is a strategy\nthat has produced successful results. The use of drug-phospholipid-oil complex in a\nliquid system was first described by  Wang  et\nal.  (2008) . They prepared a formulation containing MCT, the\nherbal medicinal compound Hydroxysafflor yellow A, phospholipid, and surfactant, and\nreported better kinetic parameters than the aqueous solution of the drug ( Wang  et al.,  2008 ). Further\nresearch has shown that drug entrapment in the phospholipid layer reduces its\nbacterial and enzymatic degradation during the absorption process ( Sun  et al.,  2013 ). The effect\nof lipids on the bioavailability of orally administered drugs is very complex,\nbecause they can change the biopharmaceutics properties of drugs by many mechanisms,\nincluding reducing the rate of gastric emptying, increasing the rate of solubility\nin intestinal fluid, and enhancement of lymphatic transport of hydrophobic drugs\nthrough lipoprotein formation. Factors such as the length of the triglyceride chain,\nthe degree of saturation, and the volume of prescribed lipid affect the absorption\nand blood-lymph distribution of the drug, which improve the absorption of the drug\nin the target tissue.\nIn this study, the effect of VD3-containing PhytoSolve on endometrial receptivity\nmarkers was investigated for the first time. Results showed that  LIF,\nβ-Integrin,  and  HOXA10  were highly expressed in\nthe VD3-containing PhytoSolve group against the VD3 group. Therefore, the hypothesis\nwas raised that encapsulating lipophilic drugs in nanometer-sized phospholipids\nmight improve the pharmacological properties of the drugs and increase their\ntherapeutic effect ( Webb  et al .,\n2012 ). During an in vivo study in rats,  Khani & Keyhanfar (2014)  showed that PhytoSolve and Phosal-based\nformulations of mebodipine are more available compared to oil solutions. Also,  Wajda  et al . (2007)  increased\nthe bioavailability of coenzyme Q10 and vitamin E through the use of PhytoSolve\n( Wajda  et al ., 2007 ).\nOne of the components of PhytoSolve is pure phospholipids. S75 is used to produce a\nwide range of drug delivery systems to improve dissolution, stability, and delivery\nof the active pharmaceutical ingredient to the target site, such as mixed micelles,\ndifferent types of liposomes, SLN, and more ( Bocca\n et al ., 2015 ). In our previous and recent study, S75\nlipoid helped to treat PCOS ( Hakimpour  et\nal ., 2022 ) and increase implantation markers. Previous\nreports showed that drug delivery based on nanoparticles reduces IC50 and improves\npharmacological activity ( van Hoogevest,\n2017 ). Other studies that formulated drugs with phosal 50PG showed that this\nmodel increased the level of the active substance in the blood and improved\ntherapeutic effects ( Fricker  et\nal ., 2010 ). In general, phospholipids increase the dissolution\nof the active substance, maintain drug dissolution in the GI tract, and improve drug\nabsorption and bioavailability ( Fricker  et\nal ., 2010 ).\nVitamin D deficiency can cause a decrease in 1,25-OHD, insulin secretion, insulin\nreceptor and an increase in insulin sensitivity, inflammation and oxidative stress.\nVitamin D deficiency can also result in a decrease in SHBG and an increase in\ntestosterone levels, leading to hyper-androgenism, hirsutism and acne. Additionally,\nvitamin D deficiency can disrupt calcium regulation, leading to halted follicular\ngrowth and lack of ovulation and infertility ( Morgante  et al ., 2022 ).\nThe results of our study also showed that VD3-containing PhytoSolve improved the\nbioavailability of VD3 and had a more positive effect on endometrial receptivity and\nexpression of genes involved in implantation than free VD3. The population included\nin this study consisted of only 36 mice; further studies with larger populations are\nneeded to determine whether the results observed in this study are valid. A\npopulation of limited size was one of the limitations of this study.\n\nBased on our results, VD3 with PhytoSolve was more successful at increasing the\nexpression of genes involved in implantation in the PCOS model than vitamin D3 in\nsuspension. Therefore, the Phytosolve formula is a reliable drug delivery method for\nmedications with limited solubility and bioavailability.","source_license":"public-domain-us","license_restricted":false}