{"paper_id":"7c29f0c3-5d5a-453a-8ed4-d08c1a2a51da","body_text":"Successful pregnancies in humans and non-human\nmammals rely on a unique set of events, such as embryo\nimplantation, separation, mating, and parturition. Implantation is associated with molecular and physiological\nevents regulated between the embryo and the receiving\nendometrium. In the implantation process in humans, fundamental events such as adhesion, adhesion / attachment,\ninvasion, and immune regulation occur ( 1 ).\nSpontaneous abortion is a significant issue in terms of\nsocial and economic effects. Today, most women face the\npossibility of reduced fertility and increased spontaneous\nabortion due to delayed pregnancy. Infertility has various\ncauses, the most common of which are tubular and pelvic\ndiseases, ovulation disorders, polycystic ovary syndrome\n(PCOS) and premature ovarian failure ( 2 ).\nInsulin is a pivotal metabolic hormone for regulating energy homeostasis in the body.\nInsulin-dependent signaling also plays an important role in embryo reproduction and early\ngrowth ( 3 ). In humans, insulin and proinsulin levels (prohormones with less activity than\ninsulin) are significantly associated with weight, height, head circumference, and skin\nthickness of infants at birth ( 4 ). Insulin sends messages through its heterotetrameric\nreceptor. After binding of insulin to alpha extracellular subunits, deformation occurs in\nthe second tyrosine kinase present in the two beta intracellular subunits, resulting in\nactivation of tyrosine kinase to auto-phosphorylate tyrosine components in the Tyr-1158,\nTyr-1162, and Tyr-1163 positions, followed by rapid phosphorylation of docking proteins such\nas insulin receptor substrates (IRS) and several other signaling proteins ( 5 ). In endometrial\ncancer, the insulin hormone, as a growth factor, can increase cell proliferation and inhibit\nthe process of apoptosis through the PI3K/Akt and RAS/MAPK pathways ( 6 ,  7 ). Activation of\ninsulin receptor ( INSR ), insulin receptor substrates -1\n( IRS-1 ) and  AKT  has also been linked to the invasive\nnature of endometrial cancer, and insulin has mitogenic and anti-apoptotic properties for\nthese cells ( 6 ).\nHuman placental growth hormone is increased continuously during the first 20 weeks of\ngestation, and this hormone has a strong effect on insulin metabolism. Because of this, the\ninsulin signaling pathway is necessary to regulate cell metabolism. In the present study, we\nhypothesized that energy balance was essential for embryo implantation and growth.\nTherefore, the disruption of the insulin signaling pathway due to decreased expression of\n INSR  and  IR-1  genes in the endometrial tissue of\ninfertile women is considered a factor affecting infertility and abortion in  in\nvitro  fertilization (IVF).\n\nIn this case-control study, two groups were selected\nfrom the clients referred to the infertility centers of Yas\nand Mirzakoochak Khan Hospitals in Tehran (2018-\n2019). Forty-two women with children, who had experienced at least one normal pregnancy, were selected as the\nfertile group. Forty-two women without children with a\nregular menstrual cycle that were married more than one\nyear and also had an unknown reason for infertility were\nselected as the infertile group. The sample size was calculated based on the following assumption: type1 and 2\nerrors: 0.05 and 0.20, respectively; expected implantation\nrate in control group: 65%; expected frequency of abortion: 35%. The infertile group underwent the IVF method\nto get pregnant, but the fertile group had a normal pregnancy. Both groups had an abortion under twenty weeks\nfor unknown reasons. The aborted fetus also had a normal\nkaryotype. The selection criteria of the groups were as\nfollows: regular menstrual cycles, normal ovarian function, and absence of abnormalities in the uterus and fallopian tubes, or signs of endometriosis on ultra-sonographic\nor laparoscopic examinations. In addition, the spouses of\nsubjects had sufficient sperm volume; and analysis of semen was according to WHO criteria. Those who did not\nhave this characteristic were excluded.\nThe subjects ranged in age from 24 to 36 years. Endometrial samples of individuals were collected using a\nNovak curette/ Pipelle catheter and transferred to a karyotype containing RNA to be stored in liquid nitrogen until\nRNA extraction.\nApproximately 150-200 mg of endometrial tissue samples were washed twice with phosphate buffered saline\n(PBS, Bioidea, IRAN). Then, the RNA of all samples was\nextracted with the help of a commercial kit instruction\n(Invitrogen, Carlsbad, CA, USA). After evaluating the\nquantity and quality of the extracted RNA according to\nthe kit instructions (Takara Bio Inc., Japan) about 1 mg\nof the total RNA from each sample was added to random\nhexamer primers, RT enzyme, and enzyme buffer used for\ncDNA synthesis and placed in a thermos-cycler.\nUsing the ABI StepOne Plus TM  system (Applied Biosystems, Germany), gene\nexpression ( INSR  and  IRS-1 ) was evaluated by\nquantitative real time polymerase chain reaction (qRT-PCR). Primers (F:\n5′-TTCCGAGACCTCAGTTTCCC-3′ and R: 5′-AGATGACCAGCGCGTAGTTA-3′) were used to proliferate the\n INSR  gene, primers (F: 5′-AGGTGGATGACTCTGTGGTG-3′ and R:\n5′-GGGATTGTTGAGATGGTGCC-3′) were used for the  IRS-1  gene, and primers (F:\n5′-CGTGCGTGACATTAAAGAGAA-3′ and R: 5′-GGGATTGTTGAGATGGTGCC-3′) were used for the\n beta-actin  gene (internal control). The proliferation steps included\n95°C for 5 minutes for initial DNA denaturation, then 35 cycles at 95°C for 30 seconds,\n55°C and 60°C for 30 seconds, and 72°C elongation for 30 seconds. All tests were performed\nin pairs. Several proliferated products were sequenced. To analyze the sample\nproliferation, the threshold line was drawn based on the exponential phase of the products\nto be statistically analyzed using the 2 -ΔΔCT  method.\nData were analyzed using Graph Pad software version 9. The normal distribution of data was\nfirst examined by the Kolmogorov - Smirnov test. Then the variables of age, BMI, duration of\nmarriage and length of pregnancy were calculated based on an independent t test and were\nreported as mean ± SD. Other data such as diabetes, number of children and abortions were\ncalculated based on Fisher’s exact test between the two groups. The expression level of\ngenes was reported as fold change according to the formula fold change=2 -ΔΔCt .\nThe fertile and infertile groups were divided into two subgroups for age (30≥ and\n30<), body mass index (BMI, 25≥ and 25<), diabetes (healthy and diabetic),\nlength of pregnancy (10 ≥ and 10< week). Fold change of the  INSR \nand  IRS-1  gene expression was compared between subgroups, using a\ntwo-sample t test. The differences in expression of INSR and IRS-1 genes in the two groups,\nthe effect of age, BMI, diabetes, and length of pregnancy on gene expression were assessed\nby t-test. The missing data were excluded from the study. In all statistic testes, a P value of\nless than 0.05 was considered significant. Results were reported with 95% confidence\nintervals (CIs).\nThe study protocol conforms to the ethical guidelines of\nthe 1975 Declaration of Helsinki as reflected in a prior approval by the Tehran Islamic Azad University of Medical\nSciences (IR.IAU.TMU.REC.1397.007). After obtaining informed consent, the structured questionnaires were\nfilled out by subjects.\n\nThere was no significant difference between the mean\nage of the two groups, duration of the marriage, number\nof abortions, smoking and diabetes. In terms of mean\nBMI, duration of pregnancy and the number of children\nthe groups were statistically significant. Individual\ninformation is presented separately in Table 1.\nIn the fertile group, the expression of the  INSR  gene was\n2.61 times higher (P=0.002, 95% CI: 0.639-2.622) and\nthe  IRS-1  gene was 2.87 times higher (P=0.008, 95% CI:\n0.177-1.137) than the infertile group. These differences\nwere also statistically significant. The results are shown\nin Figure 1.\nQuantitative real-time polymerase chain reaction (PCR) validation of transcriptome data for\n INSR  and  IRS-1  genes. The mRNA fold change was\nused for comparative gene expression between fertile and infertile women. Independent\nsamples student’s t test was performed to compare  INSR  and\n IRS-1  expression between fertile and infertile women. \n** ; P<0.001.\nBaseline characteristics of the infertile and fertile women\nData are presented as mean ± SD or n (%). Age, BMI, duration of the marriage and length of pregnancy were calculated based on the independent t test. Fisher’s exact test was used to\ncompare the distribution of other variables (abortion, diabetes and number of children) between the two groups. BMI; Body mass index and CI; Confidence intervals.\nIn terms of age, each group was divided into two\nsubgroups ≤30 and >30 years. Sixteen women in the\nfertile group and nineteen women in the infertile group\nwere ≤30 years old; and 26 in the fertile group and 23\nin the infertile group were >30 years old. In comparison\nwith the fertile group, the expression of the  INSR  gene\nwas 2.95 times (P=0.005, 95% CI: 0.397-4.010) higher\nand the  IRS-1  gene was 2.92 times (P<0.0001, 95% CI:\n0.204-1.719) higher than that of the infertile group with\nthe age of ≤30. The same comparison at age >30 showed\nthat the expression of the  INSR  gene increased by 2.42\ntimes (P=0.001, 95% CI: 0.147-2.459) and the expression\nof the  IRS-1  gene increased by 1.59 times (P=0.356,\n95% CI:-0.131-0.333). Both groups did not differ in\nthe expression of the  IRS-1  gene, except for the ≤30 age\nrange. The results are shown separately in Figure 2.\nThe effect of age parameter on the expression of  INSR  and  IRS-1 \ngenes. The mRNA fold change was used for comparative gene expression between fertile\nand infertile women. Independent samples student’s t test was performed to compare\n INSR  and  IRS-1  expression between the two age\ngroups.  ** ; P<0.001,  **** ; P<0.00001, and ns;\nP>0.01.\nTwenty-six fertile women and seven infertile ones had a\nBMI ≤25, and 16 fertile women and 35 infertile ones had a\nBMI >25. The expression of both genes was decreased by\nincreasing BMI. Comparison of BMI ≤25 in the fertile women\ncompared to the infertile women showed that the expression\nof the  INSR  gene was 10.07 times (P=0.002, 95% CI: 0.251-\n5.161) and  IRS-1  gene was 4.31 times (P<0.0001, 95% CI:\n0.533-2.270) higher. Also, fertile and infertile persons at\nBMI >25 had 1.78 times (P=0.042, 95% CI: 0.214-2.026)\nmore expression of the  INSR  gene and 2.19 times (P<0.0001,\n95% CI: 0.069-0.812) more expression of the  IRS-1  gene. A\ncomparison of the subgroups is shown in Figure 3.\nThe effect of BMI parameter on the expression of  INSR  and  IRS-1 \ngenes. The mRNA fold change was used for comparative gene expression between fertile\nand infertile women. Independent samples student’s t test was performed to compare\n INSR  and  IRS-1  expression between the BMI of the\ntwo groups of women. BMI; Body mass index,  *  ; P<0.01,\n ** ; P<0.001, and  **** ; P<0.00001.\nNine fertile women and four infertile ones had\ndiabetes (type II). Diabetes affected the expression of\ngenes and caused a reduction in the expression of both\ngenes in subjects with diabetes compared to healthy\nones. This difference was statistically significant for\nthe expression of the  INSR  gene (P=0.007) and  IRS-1 \ngene (P=0.029). Healthy fertile subjects had 23.82 times\nhigher expression of the  INSR  gene than the fertile ones\nwith diabetes (P<0.0001, 95% CI: 2.207-4.293) and\n13.83 times higher  IRS-1  gene (P<0.0001, 95% CI:\n0.679-1.813). Healthy infertile subjects showed 21.35\ntimes more expression of the  INSR  gene (P<0.0001, 95%\nCI: 0.230-0.604) than the infertile ones with diabetes\nand 16.82 times more expression for the  IRS-1  gene\n(P<0.0001, 95% CI: 0.091-0.152). The comparison of\nthe subgroups is shown in Figure 4.\nThe length of pregnancy was shorter in the infertile\ngroup than in the fertile group. This length was divided\ninto two subgroups: ≥10 weeks and <10 weeks. In the\nfertile group, the expression of the  INSR  gene was 2.79\ntimes (P<0.0001, 95% CI: 0.130-2.503) higher in the first\nten weeks of pregnancy and 3.63 times (P<0.0001, 95%\nCI: 0.697-3.071) higher in the second ten weeks than the\ninfertile group. In terms of  IRS-1  gene expression, the\nfertile group had 8.71 times (P<0.0001, 95% CI: 0.332-\n3.165) more expression in the first ten weeks and 1.48\ntimes (P=0.653, 95% CI:-1.064-0.321) more in the\nsecond ten weeks. The results of gene expression in the\nfirst ten weeks and the second ten weeks are shown in\nFigure 5.\nThe effect of diabetes on the expression of  INSR  and  IRS-1 \ngenes. The mRNA fold change was used for comparative gene expression between fertile\nand infertile women. Independent samples student’s t test was performed to compare\n INSR  and  IRS-1  expression between women who were\nhealthy or with diabetic disease.  **** ; P<0.00001 and ns;\nP>0.01.\nThe effect of the length of pregnancy on the expression of  INSR  and\n IRS-1  genes. The mRNA fold change was used for comparative gene\nexpression between fertile and infertile women. Independent samples student’s t test was\nperformed to compare  INSR  and  IRS-1  expression and\nthe length of pregnancy between groups.  *  ; P<0.01 and ns;\nP>0.01.\n\nReproduction is controlled by the common function of several neuronal and hormonal signals\n(neurohormonal system). For central reproduction controlling, the deca-peptide\ngonadotrophin-releasing hormone (GnRH) is formed to activate the lower elements of the\nhypothalamus-pituitary-gonadal (HPG) axis, especially the secretion of the famous\ngonadotrophins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Also,\nenvironmental hormones affect GnRH activity. These gonadal hormones and various metabolic\nfactors are essential for regulating energy homeostasis and fertility. Among these, insulin\nis a pivotal regulator of the HPG axis. Removing insulin receptors in animal models led to\nthe development of severe metabolic disorders, hypogonadotropism, hypogonadism, and\ninfertility ( 8 ). A study by Anjali and his colleagues demonstrated the effect of FSH on the\nexpression of genes related to energy homeostasis. They showed that FSH could increase the\nexpression of the  IRS-2  gene and the functional deficiency of FSH reduced\nfollicular growth and metabolism and led to infertility.\nThe pivotal role of the reproductive function of insulin activity in humans is determined\nby the expression of the insulin receptor in most tissues of the body, the hypothalamus,\npituitary, uterus and ovaries ( 8 ). The binding of insulin to its INSR receptor causes\ninduction of tyrosine phosphorylation in the insulin receptor substrate (IRS). Then the\nsignal is transmitted through downstream enzymes such as  PI3K  and\n AKT2 . Knockout mouse model of INSR causes hyperinsulinemia and\nhyperglycemia rapidly following diabetic ketoacidosis ( 9 ).\nHuman implantation is a complex and multifactorial\nprocess. Successful implantation requires some factors\nsuch as a healthy embryo, a receptive endometrium,\nthe molecular coordination between them, and the\nprotection of the host's immunity. Endometrial tissue has\na transient functional state and allows blastocysts to be\nimplanted and pregnancy to occur ( 10 ). Recent advances\nin the study of implantation processes have indicated that\nendometrial acceptance evaluation and pre-implantation\ngenetic testing are necessary to overcome the possibility\nof implant failure ( 11 ,  12 ) and successful initiation of\npregnancy. Early detection of endometrial abnormalities\nand the discovery of new strategies increase the chances\nof pregnancy, especially in infertile women.\nIn this study, a comparison between infertile women\nwho had undergone IVF and fertile women was made.\nBoth groups had an abortion less than twenty weeks\nfor unknown reasons. The infertile group had lower\nexpression of the  INSR  and  IRS-1  genes in uterine\ntissue compared to the fertile group. This difference of\nexpression was statistically significant. The effect of some\nvariables on gene expression was also evaluated.\nThose in each group had less gene expression with aging\n(over 30 years). This reduction was more in the fertile\ngroup than in the infertile group. Comparing infertile with\nfertile women indicated a significant relationship between\naging and the rate of decreased expression of insulin\nmessaging genes. Also, Dunson et al. ( 13 ) examined\nthe relationship between age and fertility. Their results\ndemonstrated that women aged 19-26 were significantly\nmore likely to become pregnant than women aged 27-29,\nand the infertility percent was estimated at 8% for women\naged 19-26 and 13 to 14% for women aged 27 to 34, and\n18 % for women aged 35 to 39.\nThe role of obesity is pivotal due to the increased\nproduction of hormones derived from adipose tissue,\nespecially leptin ( 14 ). Leptin plays a role in energy\nbalance and reproduction ( 8 ). Lack of leptin signaling in\nrats and humans causes obesity and infertility. Increased\nleptin in obese people reduces the activity of the\nhypothalamic-pituitary- gonadal (HPG) axis by creating a\nstate of resistance ( 14 ). In the current study, the subgroups\nwith BMI ≥25 and BMI <25 were also examined. Thirty\ninfertile individuals and only sixteen fertile individuals\nhad a BMI >25. Comparison of the two groups showed\nthat the expression of both genes is decreased by\nincreasing BMI. In obese fertile women, expression of\nboth genes decreased significantly, but the infertile group\nshowed a slight expression decrease in the  INSR  gene and\nan increased expression in the  IRS-1  gene.\nBecause insulin directly stimulates GnRH secretory activity ( 8 ), hyperglycemia occurs by\ndecreased insulin secretion in diabetes. Also, diminished insulin secretion leads to\ninfertility for reasons such as damage to the hypothalamic-pituitary-gonadal axis, increased\nDNA damage, oxidative stress, increased endoplasmic reticulum stress, mitochondrial function\ndamage, and cell pathway modulation. Regulation of insulin levels directly affects\n INSR  and  IGF1R  expression. Also, it leads to activation\nof signaling pathways associated with cell proliferation, differentiation, metabolism, and\nsurvival. In men with unexplained infertility, the lack of  INSR  and\n IGR1R  in Sertoli cells causes reduction of testicular size by 75% and\ndaily sperm production ( 15 ), insulin resistance also affects reproductive anomalies and their\nmetabolism ( 16 ). In the present study, women with diabetes in both groups had a low-level\nexpression of  INSR  and  IRS-1  genes compared to healthy\nsubjects. But comparing infertile women with diabetes with fertile women with diabetes did\nnot indicate a significant difference in terms of gene expression.\nConcerning the length of pregnancy until termination,\nthe two groups were divided into two subgroups of\nwomen less than 10 weeks pregnant and the women in\nthe second 10 weeks of pregnancy. It aimed at evaluating\nthe expression levels of  INSR  and  IRS-1  genes. The fertile\nwomen in the second 10 weeks of pregnancy showed that\nthe expression levels of  INSR  and  IRS-1  genes increase\nand decrease, respectively. Infertile women in the second\n10 weeks had a slight increase in  INSR  gene expression\ncompared to the women in the first 10 weeks. They had\na significant increase in  IRS-1  gene expression. It seems\nthat decreasing or increasing one of the genes could\ndisrupt the insulin signaling pathway.\n\nHormones affect fertility and cause changes in gene\nexpression for implantation and fetal growth through\nmessaging pathways. Disorders in the signaling pathway\nof endometrial tissue can be one of the reasons for the lack\nof fetal growth and abortion. One of the most important\nhormones is insulin, which transmits the message inside\nthe cell through the receptor and the receptor substrate.\nGenetic changes in infertile women lead to reduced\nexpression of these proteins and disrupted hormone\nsignaling. Other factors such as obesity, diabetes, old age\nand smoking also reduce the expression of these genes\nand aggravate the problem of infertility. Therefore, it\nis apparent that genetic disorders are one of the factors\naffecting infertility.","source_license":"CC-BY-4.0","license_restricted":false}