{"paper_id":"e5e832e5-ddec-4de3-b3c0-320ab590267a","body_text":"Recurrent miscarriage occurs in 1-2% of women\nwith reproductive age. Different terms and guideline\nis proposed to describe recurrent pregnancy loss. For\nexample, it is termed as “recurrent pregnancy loss\" and\n“recurrent miscarriage” by respectively the European\nsociety of human reproduction and embryology (ESHRE)\nand royal college of obstetricians and gynaecologists\n(RCOG) of UK. The new definition of ESHRE and the\nAmerican society for reproductive medicine (ASRM) for\nrecurrent miscarriage is the loss of two or more consecutive\npregnancies, whereas the earlier description was defined\nas three or more consecutive pregnancy losses ( 1 ).\nRecurrent pregnancy loss (RPL) is the most common\ncomplication of pregnancy. Almost 70% of human\nconceptions do not survive a live birth. Approximately\n50% of all pregnancies end in miscarriage before clinical\ndiagnosis even with fetal heart activity ( 2 ). RPL is\ncharacterized by two or three consecutive miscarriages\nprior to the 20th week of gestation ( 3 ). Pathogenic factors\nwhich have been known in only 50% of the cases include\nimmune, endocrine, genetic and infectious factors, as\nwell as metabolic disorders, anatomical abnormalities and\nother unknown causes ( 2 ,  3 ).\nAs a hormone secreted by the anterior pituitary gland,\ncentral nervous system, immune system, uterus, the\ntissue involved in pregnancy, and even the mammary\nglands, prolactin has several chemical forms after\ntranslation. It emerges a range of chemical changes,\nsuch as phosphorylation or glycosylation ( 4 ). It relies\non the estrogen, progesterone, glucocorticoids, insulin, thyroid hormone and parathyroid hormone. Prolactin also\nenhances uptake of some amino acids and glucose as well\nas the production of milk sugar and milk fats ( 5 ).\nProlactin is a member of the growth hormone-placental\nlactogen family, arisen from a common ancestral\ngene, about 500 million years ago. More than 300\ndifferent functions ( 6 ), including growth, development,\nreproduction, metabolism, water and electrolyte balance,\nbrain and behavior, and immune system regulation, are\nreported for prolactin ( 7 ), most of which are related to\nlactation and reproduction ( 8 ). Thus, its secretion is\nincreased during pregnancy ( 9 ). Prolactin receptors\nare located on endometrial cells and, by binding to the\nhormone, help the endometrium capability to accept\negg and create a suitable environment for blastocyst\nimplantation ( 10 ). The prolactin effects are exerted\nthrough the  CSF-1R  receptor. This receptor is a member\nof the prolactin family, belonging to the tyrosine kinase\nreceptors which are also expressed in macrophages\nand dendritic cell ancestors. This receptor controls\nproliferation, differentiation and survival of macrophages\n( 11 ). In addition,  CSF-1R  is more expressed in the cell\ncolumns of extravillous trophoblasts, anchoring placenta\nto uterus ( 12 ).\nColony-stimulating factors (i.e. M-CSF, CSF1, GMCSF, CSF2, G-CSF and CSF3) are a family of cytokines,\namong which M-CS and GM-CSF are expressed during\npregnancy in the oviduct and uterus ( 13 ).  CSF-1  (MCS) is a factor that promotes growth of immune cells,\nespecially monocytes. It is mainly produced by fibroblast\ncells, but there are various reports of its presence in the\nother tissue ( 14 ). This glycosylated homodimer with\na disulfide bond is also expressed in the endometrium,\ndecidua and placenta. In the endometrial glands, high\nlevels of  CSF-1  are seen during the secretory phase\ncompared to the proliferative phase. Decidua also shows\nhigh levels of mRNA and  CSF-1  protein in the secretory\nphase compared to the proliferative phase endometrium.\nIn addition, it seems that endometrial cells near the\nanchoring villi of trophoblast are the main sources of\n CSF-1  for the placental-uterine interface ( 12 ).\nRecurrent miscarriage and RPL mostly occur in the first\ntrimester of pregnancy, prior to the week 20 of gestation;\nin addition, abortion (28th week of gestation) or premature\nbirth (after 28th week of gestation) events occur in 10-\n20% of fertile couples ( 15 ). The current study aimed to\nevaluate prolactin hormone change and its relationship\non the expression of  CSF-1  and  CSF-1R  genes during\nmiscarriage prior to the week 20th of gestation in three\ngroups of women with infertility, RPL and healthy fertile.\n\nIn this case-control study, three groups of women\nwith infertility, RPL and healthy fertile were selected\nfrom patients referred to Yazd Infertility Center (Yazd,\nIran), as well as Yas and Mirza Kuchak Khan Hospitals\nin Tehran, Iran. Women with children who have had at\nleast two times normal pregnancy were assigned to the\nfertile group. Women with unknown causes of infertility\nand normal menstrual cycles with passing at least five\nyears from their marriages were enrolled in the infertile\ngroup. Additionally, women who passed at least five\nyears from their marriages and experienced a miscarriage\nat least twice without any children were assigned to the\nRPL group. Each group included 40 subjects (the sample\nsize was estimated based on the following assumption:\ntype1 and 2 errors: 0.05 and 0.20, respectively; expected\nimplantation rate in the control group: 65%; expected\nfrequency of abortion: 35%). Women in both infertility\nand RPL groups attempted to conceive via IVF, but\nsubjects in the fertile group had normal pregnancies. The\ninclusion criteria were as follow: having a normal ovarian\nfunction, regular menstrual cycles, normal fallopian\ntubes, lack of uterine abnormalities, lack of endometriosis\nsigns in ultrasound or laparoscopic examinations, and\nmiscarriage of unknown causes with a normal embryonic\nkaryotype prior to the week 20th of gestation. In addition,\ntheir spouses should have a normal volume and analysis\nof semen, based on the World health organization (WHO)\nreference values.\nThe selected individuals were within the age range\nof 25 and 35 years. Serum samples were taken from\nall subjects before undergoing curettage and stored at\n-20°C. Endometrial specimens were also collected using\nthe Novak curette/Pipelle catheter and stored at -20°C\nafter transferring to vials containing RNA-later. Other\ninformation about their age, height, weight and blood\npressure was extracted from their files.\nSerum prolactin concentrations were assessed by the\ncommercially available kits (REF: DKO011, LOT No.:\n4808A, DiaMetra, Italy) based on ELISA.\nFirstly, endometrial tissue samples (approximately\n100-150 mg) were rinsed with saline to remove RNA-later. Then, whole RNA was extracted from the tissue\nusing a commercially available kit (Roche Diagnostics,\nGermany) according to the manufacturer’s instructions.\nThen, the extracted total RNA was evaluated using\nspectrophotometry and gel electrophoresis. To synthesize\ncDNA, 1 mg of the total RNA of each sample was\nmixed with random hexamer primers, RT (Reverse\nTranscriptase) enzymes and enzyme buffer, according\nto the kit instructions (Gene All Inc., South Korea) and\nplaced in a thermocycler.\nBeta-actin housekeeping gene was used to evaluate\nexpression levels of  CSF-1  and  CSF-1R  genes. Sequences\nof the designed primers and length of the proliferated\nsegments are shown in Table 1.\nPrimer sequence and polymerase chain reaction (PCR) product of\n CSF-1  and  CSF-1R  genes\nSPSS version 25 software (IBM, USA) was used for data\nanalysis. The inter- and intra-group comparisons in terms of\nthe mean age, BMI and prolactin levels were performed by\none-way ANOVA and the post hoc Tukey test. The results\nwere expressed as mean ± SD. Due to the non-normality\nof the data, nonparametric Kruskal-Wallis test was used to\ncompare gene expression among the three groups. Pearson’s\ncorrelation coefficient was used to evaluate the relationship\nbetween the prolactin level and the expression level of CSF-1\nand  CSF-1R  genes. Significant level was considered P<0.05.\nThe study protocol conforms to the ethical guidelines\nof the 1975 Declaration of Helsinki as reflected in a prior\napproval by the Tehran Islamic Azad University of Medical\nScience (IR.IAU.TMU.REC.1397.007, Tehran, Iran).\nInformed consent was obtained from all participants and\ntheir information was received through a questionnaire.\n\nDemographic characteristics of the subjects in the\nfertile, RPL and infertile groups, including age, body\nmass index (BMI), smoking habit, diabetes status and\nprolactin levels, are shown in Table 2.\nMean prolactin level had a significant difference\nbetween the fertile and infertile groups (41.84 ± 4.32\nvs. 24.38 ± 1.43 ng/mL, P<0.001). The same result was\nobtained between the fertile and RPL (32.45 ± 4.16 ng/\nmL, P=0.121). The results are shown in Figure 1.\nComparison of the prolactin levels among fertile, infertile and\nmiscarriage. Comparison of the three study groups showed that the\nhighest and lowest levels of prolactin belonged to the fertile and infertile\ngroups respectively, and a significant difference was observed among\nthese groups. ***; P<0.0001 and ns; P>0.01.\nExpression level of the  CSF-1  gene in the endometrial\ntissue was higher in the fertile than the infertile group by\n2.88 times (P<0.0001). Its expression level was 2.28 times\nhigher than the RPL ones (P<0.0001). Its expression level\nbetween the RPL was 1.26 times higher than the infertile\ngroup (P=0.011).\nExpression level of the  CSF-1R  gene was 2.64 times\nhigher in the fertile group than infertile ones (P<0.0001),\nand 1.69 times higher than the RPL group. The difference\nwas statistically significant (P<0.0001). Expression level of\nthe  CSF-1R  gene in the RPL group was 1.56 times higher\nthan the infertile group, and the difference was statistically\nsignificant (P=0.002). Data are shown in Figure 2.\nCharacteristics of the fertile, infertile and recurrent abortion women\nNegative 37 (92.5) 34 (85) 32 (80)\nData are presented as mean ± SD or n (%). BMI; Body mass index.\nComparison of  CSF-1  and  CSF-1R  gene expressions between the\nstudied groups. Expression changes of the A.  CSF-1  and B.  CSF-1R  genes\namong the three study groups are shown based on the fold change.\nThe lowest and highest expression levels of the  CSF-1  and  CSF-1R  gene\nwere observed in the infertile and fertile groups, respectively. *\n; P<0.01, **; P<0.001, ***; P<0.0001, ****; P<0.00001, and ns; P>0.01.\nEffect of ≤40 and >40 ng/mL prolactin concentrations\nwas evaluated on expression level of  CSF-1  and  CSF-1R \ngenes in the fertile, infertile and RPL groups. Expression\nlevel of  CSF-1  gene in subjects with prolactin serum\nconcentration >40 ng/mL was increased 1.63, 1.01 and 1.36\ntimes in the fertile, infertile and RPL groups, respectively,\ncompared to the ones with prolactin serum levels ≤40 ng/\nmL. Differences for the infertile group were insignificant.\nExpression level of  CSF-1R  gene in the individuals with\nprolactin serum concentration >40 ng/mL was respectively\nincreased 2.86, 1.07 and 1.54 times in the fertile, infertile\nand RPL groups, respectively than those with prolactin\nserum levels ≤40 ng/mL; the differences were statistically\nsignificant unless infertile group. Data are shown in Figure 3.\nChanges in the expression levels of  CSF-1  and  CSF-1R  genes under the\ninfluence of prolactin hormone. Expression changes of the A.  CSF-1  and\nB.  CSF-1R  genes among the three study groups are shown based on the\nfold change. By increasing serum concentration of prolactin to >40 ng/mL,\nexpression level of the  CSF-1R  gene was increased in the three groups compared\nto those with prolactin serum levels ≤40 ng/mL; although the differences were\ninsignificant for infertile group. **; P<0.001, ****; P<0.00001, and ns; P>0.01.\nComparison of the subjects in the two age groups of ≤30\nand >30 years showed significant differences in the  CSF-1 \nand  CSF-1R  gene expression levels of the fertile and RPL\ngroup. In comparison, there was no significant difference\nin the expression level of genes between the infertile\nsubgroups. However in the age ≤30 of the fertile, infertile\nand RPL groups, expression level of the  CSF-1  gene in\nendometrial tissue was 1.34 (P=0.0096), 1.02 (P=0.736)\nand 1.23 (P=0.031) times higher than the age group >30\nyears. Expression level of the  CSF-1R  gene was higher 1.42 (P=0.0015), 1.33 (P=0.620) and 1.34 (P=0.031) times\nin the fertile, infertile and RPL groups, respectively.\nEvaluation of the subjects in the BMI groups ≥25\nand 25> kg/m 2 \nshowed intragroup differences for the\nexpression of the  CSF-1  and  CSF-1R  genes. In other\nwords, expression level of the  CSF-1  and  CSF-1R  genes\nwas decreased with increasing BMI. Comparison of the\nsubjects with a BMI ≥25 kg/m 2 \nshowed that  CSF-1  gene\nexpression level was 1.58 (P=0.006), 1.25 (P=0.032) and\n1.48 (P=0.049) times higher than the fertile, infertile and\nRPL groups, respectively. Additionally, comparison of the\nindividuals with BMI ≥25 kg/m 2 \nshowed that expression\nlevel of  CSF-1R  was 2 (P<0.0001), 1.20 (P=0.048) and\n1.33 (P=0.034) times higher than the fertile, infertile and\nRPL groups, respectively.\nIntragroup comparisons showed significant differences\nbetween diabetic and non-diabetic subjects in each group\nby evaluating the  CSF-1  and  CSF-1R  gene expression\nlevels. Comparison of the diabetic subjects with non-diabetic individuals showed that  CSF-1  expression\nlevels were decreased 2.22 (P=0.0004), 1.58 (P=0.0073)\nand 2.13 (P=0.0002) times in the fertile, infertile, and\nRPL groups respectively. Comparison for the  CSF-1R \nexpression levels were decreased 1.42 (P=0.0028), 1.33\n(P=0.029) and 1.34 (P=0.019) times in the fertile, infertile\nand RPL groups, respectively.\nIntragroup comparisons between smokers and non-smokers showed significant differences. Expression levels\nof  CSF-1  genes in smoker subjects were decreased 1.58\n(P=0.017), 1.25 (P=0.045) and 1.49 (P=0.026) times in the\nfertile, infertile and RPL groups, respectively, compared\nto their counterparts with non-smokers. Expression levels\nof CSF-R1 genes in the smoker subjects were 2 (P=0.039),\n1.20 (P=0.025) and 1.33 (P=0.030) times lower than the\nnon-smokers who counterparts in the fertility, infertility\nand RPL groups respectively.\nIntragroup comparisons between gestational age ≤10 and\n>10 weeks showed no significant difference in the gene\nexpression levels of the infertile and PRL groups. The\nfertile group with a history of miscarriage, less than ten\nweeks, was compared to those with a history of miscarriage\nmore than ten weeks.  CSF-1  and  CSF-1R  gene expression\nlevels were increased 1.2 (P=0.002), and 1.16 (P=0.012)\ntimes, respectively. All data are shown in Figure 4.\nPearson correlation test for correlation between prolactin\nand  CSF-1  gene expression showed that the fertile group\nhad a positive correlation (r=0.64, P=0.0017), the infertile\ngroup had no significant correlation (r=0.052, P=0.575)\nand the RPL group had statistically significant positive\ncorrelation (r=0.415, P=0.008). The same study, performed\nfor the  CSF-1R  gene, showed that this correlation was\nonly seen in the fertile group (r=0.452, P=0.015), but there\nwas not any association between the infertile (r=0.098,\nP=0.285) and RPL groups (r=0.167, P=0.067).\nChanges in the expression levels of  CSF-1  and  CSF-1R  under the\ninfluence of different parameters. Effect of age, BMI, diabetes, smoking,\ngestational age and prolactin level on expression level of the studied\ngenes was observed in the current study. BMI; Body mass index, *\n; P<0.01, **; P<0.001, ***; P<0.0001 and ns; P>0.01.\n\nMany factors can contribute to the success of IVF-ET in\nfertilization and embryo transfer. The main independent\nvariables are age of the women, serum concentration of\nthe anti-Mullerian hormone, number of the transferred\nembryos and their qualities. Some researchers showed\nthat growth factors, hormones and cytokines, produced\nby macrophage cells, were involved in the implantation\nprocess ( 16 ).\nDuring pregnancy, prolactin rises above the normal level\nof 10-25 ng/mL and reaches to a peak of 200-400 ng/mL\nwithin eight weeks of gestation ( 17 ). In the current study,\nmean prolactin serum concentration was determined in\npregnancy losses at eight weeks of gestation as 41.84,\n24.38 and 32.45 ng/mL for respectively the fertile,\ninfertile and RPL groups; but in pregnancies losses <20 weeks of gestation, expression levels of  CSF-1  gene in\nsubjects with prolactin serum concentration >40 ng/mL\nwas increased 1.63, 1.01 and 1.36 times; additionally,\nexpression levels of  CSF-1R  gene were 2.86, 1.07 and 1.54\ntimes in the fertile, infertile and RPL groups respectively,\ncompared to the individuals with prolactin serum levels\n≤40 ng/mL indicating that prolactin serum concentration\nwas increased by gestational age and it affects the embryo\nsurvival. Findings showed that increasing concentration\nof serum prolactin in this group was not sufficient for the\nsurvival of embryo. It also contributes to pregnancy loss.\nMoreover, investigating the role of prolactin biomarker\nconfirmed its effect on fertility.\nIt is expected that maternal prolactin serum concentration\nis significantly elevated from 10 to 20 weeks of gestation\n( 17 ). Although the highest and lowest average serum\nprolactin levels were significantly detected between the\nfertile and infertile groups, no significant elevation was\ndetermined between the fertile and PRL groups.\nIn pregnant women, elevated levels of  CSF-1  and  CSF-1R \nexpression were observed in the endometrial epithelium\nand fetal trophoblast, respectively. Studies showed\nthat activation of trophoblast  CSF-1R  and increased\nlevel of local  CSF-1  expression were essential for the\nimplantation of a normal fetus and placental development\n( 18 ). Prolactin also affected endometrial tissue through\nthe  CSF-1R  ( 10 ). In the studied subjects, expression level\nof  CSF-1  and  CSF-1R  genes in endometrial tissue, as well\nas the gene expression changes under the influence of\nprolactin concentration, were compared among the fertile,\ninfertile and RPL groups. Expression level of the  CSF-1 \nand  CSF-1R  genes was higher in the endometrial tissue\nof the fertile group rather than the infertile ones. With\nincreasing prolactin serum levels, expressions of  CSF-1 \nand  CSF-1R  were increased in the three groups. In the\nendometrial tissue of infertile group, expression levels of\n CSF-1  and  CSF-1R  were lower than those of the fertile\nand RPL groups. Increased prolactin serum levels did not\nchange expression of the  CSF-1  and  CSF-1R  genes in the\ninfertile group. Increasing prolactin serum concentration\ncaused increasing  CSF-1  and  CSF-1R  expression levels in\nthe fertile women.\nA recent study showed that  CSF-1  expression level\nincreased up to 1000 times during gestation in the\nendometrium of pregnant mice, due to its synthesis in\nthe uterine lumen and glandular secretory epithelium\ncontrolled by the maternal endocrine hormones. Increasing\nlocal  CSF-1  synthesis in the uterus was associated with\nproliferation and differentiation of cells. Additionally,\nassociation of  CSF-1  receptor was determined between\nthe uterus and placenta for implantation.  CSF-1  also\nplayed role in regulating processes which are essential for\nimplantation and preimplantation ( 19 ). A study, performed\nby Cai et al. ( 13 ), indicated that increasing M-CSF\naffected fetal development and increased trophectoderm\n(TE) cell count in mice.\nPregnancy loss risk factors are age, weight and general health status of mother. Risk of spontaneous miscarriage\nwas increased by increasing maternal age ( 20 ). In the\npresent study, in addition to prolactin serum levels, other\nparameters, such as age, BMI, diabetes, smoking habits,\nand gestational age,  CSF-1  and  CSF-1R  expression\nwere evaluated in the all three groups. Intragroup\ncomparisons showed significant difference between the\nsubjects, aged > 30 and ≤30, in terms of gene expression\nexpected the infertile group. But, expression level of the\nboth genes was decreased by increasing age in the fertile\nwomen and those with RPL. Intragroup comparisons\nshowed a significant difference between the subjects\nwith BMI ≥25 and <25. Additionally,  CSF-1  and  CSF-1R  gene expressions were decreased in three groups by\nincreasing BMI.\nIntragroup comparisons showed a significant difference\nin the gene expression pattern between diabetic and non-diabetic subjects. However, expression level of the both\ngenes was reduced in diabetic individuals. In addition,\nthe difference was statistically significant. Intergroup\ncomparisons showed significant differences between the\nsmokers and non-smokers in gene expression levels. In\nterms of gestational age, expression of the both genes had\na significant increase in the fertile women who lost their\npregnancies at -20 weeks of gestation, but the infertile\nand PRL groups had no significant changes.\n\nOverall, hormones, such as prolactin, are among the\ndevelopment factors of fetus, which can help fetal growth\nand successful continuation of pregnancy, through affecting\nthe genes involved in conceptus–endometrial interactions.\nDecreased expression of  CSF-1  and  CSF-1R  can be\nconsidered as disruptive factors of implantation and fetal\ngrowth, leading to miscarriage in the studied groups. Factors,\nsuch as aging, increased BMI, smoking and diabetes, also\ncaused decreasing changes in gene expression ( CSF-1 \nand  CSF-1R ). It seems that decreasing expression level of\nthese genes disrupted conceptus-endometrium interaction,\nresulting in miscarriage. For future investigations, it\nis suggested to examine expression of the other genes\ninvolved in embryo implantation and their functional role\nin recurrent pregnancy loss. Additionally, discovery of the\nother molecular factors, involved in implantation, lead to\nincrease in fertility success.","source_license":"CC0","license_restricted":false}