{"paper_id":"34e07cd6-0117-4017-9a91-afc66894221f","body_text":"The COVID-19 pandemic, instigated by the SARS-CoV-2 virus, has become one of the most\nsignificant public health crises in contemporary history. Although the respiratory\nimplications of the virus are well-documented, emerging research indicates that\nCOVID-19 may also have repercussions for reproductive health [ 1 ][ 2 ][ 3 ][ 4 ]. The\nvirus gains entry into host cells through the angiotensin-converting enzyme 2 (ACE2)\nreceptor, which is present in several non-respiratory tissues, including the testes,\novaries, and placenta [ 5 ][ 6 ]. This presence raises alarms regarding the\npotential negative effects of COVID-19 on fertility and pregnancy outcomes. Numerous\nstudies have indicated the presence of SARS-CoV-2 in various female reproductive\ntissues, including the ovaries, placental tissues, amniotic fluid, and breast milk\nof infected individuals [ 7 ][ 8 ][ 9 ].\nThis indicates that the virus may spread to the female reproductive system.\nAdditionally, studies reveal elevated inflammatory cytokines in the serum and\nfollicular fluid of women with COVID-19 who are undergoing in-vitro fertilization\n(IVF) [ 10 ][ \n11 ][ 12 ]. Excessive inflammation\ncaused by COVID-19 infection may hinder oocyte quality and fertility potential, as\nsuccessful reproduction relies on a healthy ovulatory process and a well-balanced\nfollicular microenvironment [ 13 ][ 14 ].\nThe female ovary is a complex organ that contains follicles at various stages of\nmaturation. Each menstrual cycle involves the recruitment and selection of dominant\nfollicles, culminating in the ovulation of a mature oocyte. This intricately\nregulated process relies on specific interactions among ovarian follicular cells,\nneuroendocrine signals, paracrine factors, and the developing oocyte [ 15 ].\nGranulosa cells that line the follicles and the oocyte they surround engage in\nbidirectional communication to synchronize essential processes such as cell\nproliferation, differentiation, and apoptosis necessary for folliculogenesis.\nDisruptions in the molecular interactions between granulosa cells and oocytes can\nadversely affect the maturation and quality of the oocyte [ 16 ]. Previous studies have reported changes in the expression\nof inflammatory cytokines, growth factors, metabolic enzymes, and cell cycle\nregulators in the granulosa cells and oocytes of women with specific chronic\nconditions and obesity. These molecular changes were associated with impaired oocyte\ncompetence and reduced fertility outcomes [ 17 ].\nSince COVID-19 causes a pathogenic infection with significant inflammatory response\n[ 18 ], it is likely that the disease alters\nthe ovarian transcriptome too. Identifying the genes that are abnormally expressed\nin granulosa cells and oocytes could shed light on the mechanisms behind\nreproductive issues caused by COVID-19. This study aimed to thoroughly assess key\ngenes involved in inflammatory, vascular, sex hormones, and cell cycle pathways in\ngranulosa cells from women with COVID-19 undergoing assisted reproductive technology\n(ART), compared to healthy controls.\n\nFigure  1 . The mRNA levels of\ninflammatory cytokines TNF-α (A), IL-1β (B), IL-6 (C), and IL-8 (D). The\nresults of the RT-PCR analysis unveiled a significant upregulation of\ninflammatory mediators including TNF-α, IL-1β, and IL-6 in the granulosa\ncells of COVID-19 positive patients. The results are expressed as the\nmean±standard deviation (SD). Statistical significance is indicated as ****\nfor P<0.0001, *** for P<0.001, ** for P<0.01, and * for P<0.05.\nTNF-α: tumor\nnecrosis factor-alpha, IL: interleukin.\nStudy Participants\nThis study involved 30 women receiving assisted reproductive technology (ART)\ntreatment split into two groups: a study group of 15 women who tested positive for\nCOVID-19 after ovarian stimulation, and a control group of 15 healthy women matched\nby age. Before beginning ovarian stimulation protocols, all ART candidates should\nnot have a SARS-CoV-2 positive test.\nThis SARS-CoV-2 infection should also be checked on the day of the final oocyte\nmaturation triggering prior to oocyte retrieval. Some patients have a positive\nSARS-CoV-2 test when ovary is stimulated and in the day of oocyte retrieval.\nTreatment is discontinued for these patients due to potential risks for embryo.\nOocyte retrieval is advised, although, in a small number of individuals who are\nsusceptible to developing severe ovarian hyperstimulation syndrome (OHSS) and its\nassociated consequences, as well as in certain unique situations like endometriosis\nor breast cancer patients. Based on the current recommendations in infertility\ntreatment centers, the collected follicular fluid and the cells from the OHSS\npatients are discarded [ 9 ]. For this\ninvestigation, unused samples from women infected with COVID-19 were gathered. Prior\nto the trial, patients were informed verbally and in writing, given enough time to\nthink over their involvement, and provided their written consent. The Shiraz\nUniversity of Medical Sciences Scientific and Ethics Committee gave its clearance to\nthis study (approval code: IR.SUMS.REC.1400.672).\nStimulation and Granulosa Cells Collection\nFor every patient, a conventional GnRH antagonist protocol was followed. Following a\ntransvaginal ultrasound (TVU) scan on the second day of the menstrual cycle, ovarian\nstimulation was initiated with HMG (PDHoMoG®, Pooyesh Daroo, Tehran, Iran) and\nFollitropin Alpha (Cinal-F®, CinnaGene, Alborz, Iran). On cycle day six, or when the\nleading follicle reached 12 mm, the GnRH antagonist (Cetrotide®, Injection, Powder,\n250 μg, Serpero pharmaceutical, Italy) was started at a dose of 250 μg per day.\nThe drugs were kept up until the diameter of at least two follicles measured 17-18\nmm. Subcutaneous injection of 2-3 ampules of Varian Pharmed’s Variopeptyl®,\nInjection, 0.1 mg, Tehran, Iran, a GnRH agonist, was then administered to initiate\nthe last stage of oocyte maturation. Oocyte retrieval guided by TVU was carried out\napproximately 34-36 hours following triggering. Every patient’s excited follicle was\nrecovered. Following a routine isolation process, the acquired follicular fluid was\nsent to the embryology lab in order to extract granulosa cells.\nRNA Isolation and Gene Expression Analysis\nGranulosa cells were processed immediately after collection to maintain RNA\nintegrity. A commercially available RNA extraction kit was used for isolation. The\nquality and quantity of the extracted RNA were evaluated using spectrophotometry and\ngel electrophoresis. Only samples with high purity (A260/A280 ratio>1.8) and\nintact RNA profiles were chosen for further analysis. The isolated RNA was then\nconverted into complementary DNA (cDNA) using a reverse transcription kit. The\nanalysis concentrated on several key genes related to inflammation (IL-1B, TNF-α,\nIL-6, and IL-8), vascular function (VEGF and ANGPT1), cell cycle regulation (FOXL2,\nCyclin D2, Cyclin D1, and KLF4), and gonadotropin receptor signaling (luteinizing\nhormone receptor (LHCGR) and follicle-stimulating hormone receptor (FSHR)).\nAfter conducting qRT-PCR, the fold changes in gene expression between the study group\n(women with COVID-19 positive test) and the control group (healthy individuals) were\ndetermined using the 2-ΔΔCt method.\nThe data were normalized against the expression of the housekeeping gene GAPDH.\nStatistical Analysis\nData were analyzed using SPSS (version 24, IBM, Chicago, IL) and GraphPad Prism\n(version 8). Differences in baseline characteristics between the study and control\ngroups were evaluated with an independent samples t-test for continuous variables\nand a chi-square test (or Fisher’s exact test when appropriate) for categorical\nvariables, with a p-value of less than 0.05 considered statistically significant.\nGene expression differences between the COVID-19 positive group and the control group\nwere compared using the Mann-Whitney U test for non-normally distributed data or the\nStudent’s t-test for normally distributed data.\nThe relationship between comorbidities and gene expression levels was examined using\nPearson’s or Spearman’s correlation coefficients, depending on the distribution of\nthe data.\n\nFigure  2 . The mRNA levels of vascular\nmarkers ANGPT1 (A), and VEGF (B). The results of the RT-PCR analysis\nunveiled a significant upregulation of vascular markers including ANGPT1 and\nVEGF in the granulosa cells of COVID-19 positive patients. The results are\nexpressed as the mean±standard deviation (SD). Statistical significance is\nindicated as **** for P<0.0001, *** for P<0.001, ** for P<0.01, and\n* for P< 0.05. ANGPT1: Angiopoietin 1, VEGF: Vascular endothelial growth\nfactor.\n1. Study Participants\nThe study group consisted of 15 women who had tested positive for COVID-19 after\novarian stimulation, with ages ranging from 26 to 38 years and a mean age of 31.2\nyears. The control group comprised 15 healthy women matched by age who were\nundergoing ART between 27 to 36 years old with the mean age of 32.1. There was no\nstatistically difference between groups regarding the mean age.\n2. Gene Expression in Granulosa Cells\n2.1. Inflammatory Cytokines\nThe mRNA levels of inflammatory cytokines IL-1β, TNF-α, and IL-6 were significantly\nelevated in the granulosa cells of women with COVID-19 compared to the control\ngroup. Specifically, IL-1β expression was 1.4 times higher (P<0.0001), TNF-α was\n2.2 times higher (P<0.0001), and IL-6 was 1.6 times higher (P<0.01). Although\nIL-8 expression also increased, this change did not achieve statistical significance\n(Figure- 1 )\n2.2. Vascular Genes\nBoth the VEGF and ANGPT1 genes were found to be overexpressed in the granulosa cells\nof the study group. Specifically, VEGF mRNA levels were 1.6 times higher (P<0.0001),\nwhile ANGPT1 levels were 1.2 times higher (P=0.001) compared to the control group\n(Figure- 2 ).\n2.3. Cell Cycle\nThe regulators FOXL2, a vital transcription factor for granulosa cell\ndifferentiation, exhibited a downregulation in women positive for COVID-19 (P<0.0001).\nIn contrast, the cell cycle promoters Cyclin D2 and Cyclin D1 were upregulated by\n1.4-fold (P=0.0001) and 1.4-fold (P=0.001), respectively. No significant change was\nobserved in KLF4 expression (Figure- 3 ).\n2.4. Gonadotropin Receptor\nThe expression of both LH (LHCGR) and FSH (FSHR) receptor genes in granulosa cells\nwas similar between the study and control groups, showing no significant differences\n(Figure- 4 ).\n\nFigure  3 . The mRNA levels of cell cycle\nmarkers FOXL2 (A), CycD2 (B), CycD1 (C), and KLF4 (D). The results of the\nRT-PCR analysis unveiled a significant upregulation of FOXL2 and a\nsignificant downregulation of cell cycle markers including CycD2, and CycD1\nin the granulosa cells of COVID-19 positive patients. The results are\nexpressed as the mean±standard deviation (SD). Statistical significance is\nindicated as **** for P<0.0001, *** for P<0.001, ** for P<0.01, and * for P<\n0.05. Cyc: cyclin, KLF4: Krüppel-like factor 4.\nFigure  4 . The mRNA levels of vascular\nmarkers LHR (A), and FSHR (B). The results are expressed as the\nmean±standard deviation (SD). Statistical significance is indicated as ****\nfor P<0.0001, *** for P<0.001, ** for P<0.01, and * for P<0.05. LHR: LH\nreceptor, FSHR: FSH receptor.\nThe findings of this study reveal significant alterations in the molecular profiles\nof granulosa cells obtained from women who tested positive for COVID-19 compared to\nhealthy controls. Notably, there were significantly increased mRNA levels of\ninflammatory cytokines IL-1β, TNF-α, and IL-6 in the granulosa cells of women\ninfected with SARS-CoV-2 undergoing ART. This aligns with earlier findings that\nCOVID-19 infection triggers a strong inflammatory response marked by the release of\ncytokines [ 19 ]. Inflammatory cytokines such\nas IL-1β and TNF-α are known to disrupt ovarian function through various mechanisms,\nincluding inducing apoptosis in granulosa cells, interfering with steroidogenesis,\nand hindering oocyte maturation [ 20 ]. The\nincreased levels of cytokines observed in this study suggest a pro-inflammatory\novarian microenvironment in women with COVID-19, which may negatively affect\nfolliculogenesis and oocyte quality. Interestingly, although IL-8 exhibited a trend\ntoward upregulation, it did not achieve statistical significance, indicating a\ndifferential regulation of cytokines in response to SARS-CoV-2 infection.\nAdditionally, the vascular genes VEGF and ANGPT1 were found to be overexpressed in\nthe granulosa cells of women who tested positive for COVID-19. VEGF plays a crucial\nrole in promoting angiogenesis during follicle development by facilitating the\nformation of new blood vessels [ 21 ].\nIncreased levels of VEGF indicate heightened vascular permeability and angiogenesis,\nwhich may disrupt the blood-follicular barrier. The overexpression of ANGPT1, a\ncrucial mediator of vascular stabilization, protection, and remodeling, indicates\nthat significant vascular changes are occurring in response to SARS-CoV-2 infection.\nDysregulated angiogenesis and vascular instability are known to negatively impact\noocyte maturation [ 22 ][ 23 ]. Furthermore, the study found that FOXL2, a master\nregulator of granulosa cell differentiation, was downregulated, while cell cycle\ngenes Cyclin D1 and D2 were upregulated in the granulosa cells of women infected\nwith COVID-19. FOXL2 is essential for maintaining granulosa cell identity and\ncoordinating folliculogenesis with proliferation signals [ 24 ][ 25 ]. Aberrant\nexpression of FOXL2, coupled with excessive cell cycling, may result in failed\ndifferentiation and inadequate coordination of growth with developmental signals,\nultimately hindering follicle development. This study is the first to report changes\nin key cell cycle mediators in relation to COVID-19 infection, offering new\nmechanistic insights. Notably, there were no differences in the gonadotropin\nreceptor genes LH and FSH between the groups, suggesting that SARS-CoV-2 infection\nmay not directly affect pituitary-ovarian communication. However, the\ndisproportionate changes observed in other molecular regulators underscore the\ncomplex interactions among the inflammatory microenvironment, intra-ovarian\nsignaling networks, and oocyte quality in the context of COVID-19.\nA limitation of this study was its cross-sectional design, which prevents the\nassessment of long-term or recurring effects of COVID-19 on ovarian function and\nfertility potential over time. However, the current data provide new insights into\nhow SARS-CoV-2 infection disrupts various ovarian pathways at the molecular level\nduring the acute phase. By integrating clinical parameters of participants with gene\nexpression patterns, a comprehensive profile analysis was achieved. Future\nprospective studies are needed to evaluate ovarian reserve markers and the number\nand quality of oocytes following infection.\n\nIn conclusion, this research significantly enhances our understanding of the\nrelationship between COVID-19 and female reproductive health. It reveals that\nSARS-CoV-2 dysregulates expression of inflammatory cytokines, growth factors, and\ncell cycle regulators persists in the granulosa cells, indicating long-term\npathological changes. This molecular evidence supports the hypothesis that COVID-19\ninfection may disrupt follicular development and oocyte maturation through altered\nsignaling within the ovary. The findings shed light on potential pathogenic\nmechanisms and have implications for counseling and managing fertility in women who\nhave recovered from SARS-CoV-2. Further exploration of mitochondrial function, DNA\nrepair capacity, meiotic resumption, and embryo development post-fertilization could\nprovide deeper insights. Additionally, this study underscores the necessity to\ninvestigate therapeutic interventions, such as anti-inflammatory strategies, aimed\nat reversing COVID-19-related ovarian changes and optimizing fertility preservation\nin women of reproductive age who have been infected.\n\nNone.","source_license":"CC-BY-4.0","license_restricted":false}