{"paper_id":"e3cac5c7-e3e4-420a-b6bf-e6b78a22d1b4","body_text":"The coronavirus disease-2019 (COVID-19), caused by the severe acute\nrespiratory syndrome coronavirus 2 (SARS-CoV-2) is now spread all over the\nworld [ 1 ][ \n2 ][ 3 ]. The disease has major\neffects on the human life aspects like\neducation, employment, mental and physical health of individuals, and the\nworld’s economy [ 4 ][ 5 ][ 6 ][ 7 ]. The main medical societies in the field of\nreproductive\nmedicine suggested suspending the beginning of reproductive treatments or\nperforming assisted reproductive technologies (ART) with special considerations\nfor ovulation induction, in vitro-fertilization, intrauterine inseminations,\noocyte and sperm cryopreservation, as well as fresh/frozen embryo transfers [ 8 ][ 9 ].\nHowever, ART services must return to operation, especially in certain cases\nwhere postponing treatment could be more harmful than proceeding, such as\noncological or low ovarian reserve conditions [ \n8 ][ 10 ]. Many studies have shown\nthat viruses can infect the mammalian oocyte and affect the development of the\npreimplantation embryo [ 11 ][ 12 ][ 13 ].\nSARS-CoV-2 can infect several tissues and\norgans. SARS-CoV-2 infects tissues by using angiotensin-converting enzyme 2\n(ACE2) and CD147 or Basigin (BSG) as a receptor on the host cells [ 14 ][ 15 ].\nThe\nmRNA expression of these genes is demonstrated in most of the human female\nreproductive tract cells [ 16 ], endometrium\n[ 17 ], and the human embryo during\nthe early developmental stages [ 18 ]. Human\noocytes and granulosa cells also\nexpress ACE2 and BSG genes, and the corresponding proteins are present on the\nmembrane of these cells [ 19 ]. Barragan  \net al.\n  reported that SARS-CoV-2\nviral RNA was not detectable in the oocytes analyzed from two women. However,\ninfection with the SARS-CoV-2 cannot be completely ruled out, especially\nbecause of the low sample size of the study [ \n20 ]. Hypothetically, blood flowing\nthrough capillaries could be the source of infection in ovarian follicles,\nfollicular cells including granulosa-theca cells, and the oocyte [ 19 ]. Hence,\nthis study aimed to investigate the presence of SARS-CoV-2 RNA in follicular\nfluid, granulosa cells, and oocytes of COVID-19-infected women undergoing ART.\n\nFigure  1 . Hazrate zeinab hospital ovum\npick-up (OPU) flow chart.\nFigure  2 . Ghadir Mother and child\nhospital ovum pick-up (OPU) flow chart.\nThis cross-sectional study was performed on the harvested oocytes,\ngranulosa cells, and follicular fluid obtained from COVID-19-infected women.\nAccording to the instructions of the Ministry of Health and Medical Education\n(MOHME) of Iran, all the patients who are a candidate for ART during the\nCOVID-19 pandemic should have a negative nasopharyngeal COVID-19 polymerase\nchain reaction (PCR) test just before starting ovarian stimulation protocols,\nand also the test should be repeated on the day of final oocyte maturation\ntriggering before oocyte retrieval. In a small number of patients, the\nSARS-CoV-2 RNA test becomes positive after ovarian stimulation and on the day\nof oocyte retrieval. Because of great uncertainties regarding the possibility\nof vertical transmission and the negative effects of the infection on the\nembryo, treatment is stopped in these patients. However, in a few cases who are\nprone to develop severe ovarian hyperstimulation syndrome (OHSS) and its\nrelated complications, or in some special cases such as endometriosis or breast\ncancer patients, oocyte retrieval is suggested. According to the current\ninstructions in infertility treatment centers, the obtained follicular fluid\nand the cells from the OHSS patients are discarded. These unused collected\nsamples from COVID-19-infected women were investigated for the presence of\nSARS-CoV-2 RNA in their follicular fluid, granulosa cells. Patients were given\noral and written information, ample time to consider their participation, and\nconsented in writing before the study. Finally, four SARS-CoV-2 PCR-positive\npatients entered the study. This study was approved by the Scientific and\nEthics Committee of Shiraz University of Medical Sciences (approval code:\nIR.SUMS.REC.1399.1214).\nA standard GnRH antagonist protocol was carried out for all of the\npatients. On the second day of the menstrual cycle, after performing a\ntransvaginal ultrasound (TVU) scan, ovarian stimulation was started by\nFollitropin Alpha (Cinal-F®, CinnaGene, Alborz, Iran) and HMG (PDHoMoG®,\nPooyesh Daroo, Tehran, Iran). GnRH antagonist (Cetrotide®, Injection, Powder,\n250 μg, Serpero pharmaceutical, Italy) at a dose of 250 μg per day was started\non cycle day six or when the leading follicle reached 12 mm. The medications\nwere continued until at least two follicles reached 17-18 mm in diameter. Then\n2-3 ampules of GnRH agonist (Variopeptyl®, Injection, 0.1 mg, Varian Pharmed,\nTehran, Iran) were injected subcutaneously to trigger the final oocyte\nmaturation. TVU-guided oocyte retrieval was performed about 34-36 hours after\ntriggering. All of the stimulated follicles of patients were retrieved. The\nobtained follicular fluid was transferred to the embryology laboratory to\nisolate granulosa cells and oocytes based on a standard isolation protocol. To\nensure that there was no blood contamination in the follicular fluid, it was\nevaluated precisely during the isolation of oocytes and granulosa cells by\nmicroscope regarding the presence of blood cells. Figure- 1\n  and - 2  show the flow of patients and\ndetailed information about the study participants.\nThe SARS-CoV-2 test was performed for all women who were\ncandidates for ovum pick-up before starting ovarian stimulation and on the day of\ntriggering. COVID-19 laboratory confirmation was defined as a positive result\nfor SARS-CoV-2 RNA in real-time reverse transcriptase-PCR (RT-PCR) assay on\nnasopharyngeal swabs. To perform the SARS-CoV-2 test, all patients were\nreferred to laboratories approved by the Shiraz University of Medical Sciences.\nTotal RNA was extracted from follicular fluid, scrapped granulosa\ncells, and oocyte samples using the BehPrep Viral Nucleic Acid Extraction kit\n(BGene Co, Iran) according to the manufacturer's protocol. The BehPrep Viral\nNucleic Acid Extraction is designed and optimized to extract a small amount of\nviral RNA from cells, bronchoalveolar lavage, nasal and pharyngeal swabs,\nrespiratory\ndischarge, viral transfer media, and body fluids. The oocytes and granulosa\ncells were isolated from the follicular fluid, the cell-free follicular fluid\npooled, and RNA extraction was performed directly on 0.2 ml of it. No\nconcentrating steps or ultracentrifugation was performed. The collected\ngranulosa cells of each subject were pooled, and the RNA was extracted from\nthem and used for real-time PCR. RNA extraction on the oocytes of one subject\nwas performed in a pool; for two subjects, it was performed on a single oocyte\n(because they donated only one oocyte), and for the other, it was performed on\ntwo donated embryos. Real-time PCR was carried out on an Applied Biosystems\n7500 System (California, USA) using COVID-19 One-Step RT-PCR Kit (PishtazTeb\ndiagnostics, Tehran, Iran). The primer-probe of this kit adopts the dual-target\ngene design, which targets the specific conserved sequence encoding the RdRp\n(RNA-dependent RNA polymerase) region and the nucleocapsid protein N region.\nThe PCR detection system includes an internal control primer probe (RNase P) to\navoid false-negative results. The result of internal control provides the\nsampling and extraction process accuracy. The minimum detection limit of the\nkit was 200 copies/mL. The interpretation of the results was performed based on\nthe manufacturer's instructions. Real-time PCR was performed in technical\ntriplicates.\n\nFour women who had positive COVID-19 PCR nasopharyngeal tests on\nthe day of oocyte triggering and were candidates for ovum pick-up as protocol,\nwere included in this study. All the patients were asymptomatic. After specimen\ncollection, the SARS-CoV-2 RNA detection test was performed on the obtained\nfollicular fluid, granulosa cells, oocytes, and four embryos of patients. The\nresults\nof the internal control verified the accuracy of RNA extraction in the\nexperiments (the Ct of RNase P was lower than 35 and was compatible with the\nmanufacturer’s quality control instruction). Complies with the real-time PCR\nkit quality control, the Ct of negative controls were not detectable, and the\nCt of positive controls was less than 35. SARS-CoV-2 RNA was detected in one\nfollicular fluid sample (Ct less than 40 for both RdRp and N genes in\ntriplicates). The other follicular fluid samples were negative for the virus.\nFurthermore, SARS-CoV-2 RNA was not detectable in all samples of oocytes and\ngranulosa cells.\n\nTo our knowledge, up to now, there is no published study on the detection of\nSARS-CoV-2 RNA in the follicular fluid from COVID-19 PCR-positive women undergoing\nART. It is no evidence whether SARS-CoV-2 can enter and infect the human gametes and\nhow can it possibly affect the development of the human embryo [ 21 ].\nResults of the present study indicated that SARS-CoV-2 could enter follicular fluid\nas demonstrated by a positive SARS-CoV-2 RNA test in one of our four follicular\nfluid samples. However, no viral RNA was detectable in the granulosa cells and\noocytes. Since the detection limit of the kit used for viral RNA was 200 copies per\nml, the presence of the virus with fewer copy numbers cannot be definitively ruled\nout in the other tested samples. Data from the literature suggests that viruses such\nas human hepatitis B virus (HBV) [ 22 ][ 23 ], herpes simplex virus [ 24 ], Hobi-like virus [ 25 ], and porcine circovirus type 2 [ 26 ][ 27 ] can infect the\noocyte and affect the development of the pre-implantation embryo. Therefore, the\npresence of SARS-CoV-2 in the follicular fluid might probably induce negative\neffects on the oocyte and embryo development.\nData on SARS-CoV-2 presence in human follicular fluid, granulosa cells, and oocytes\nof infected individuals are poorly available to date. To our knowledge, there is\nonly one published study on the detection of the viral RNA of SARS-CoV-2 in oocytes\nfrom women who were positive by PCR, in which the viral RNA for gene N was\nundetectable in all 16 oocytes tested from two COVID-19-positive women [ 20 ]. Nonetheless, no conclusion can be obtained\nas the number of oocytes analyzed (a total of 16 oocytes) was small and only in two\nwomen. Furthermore, the patients were asymptomatic, and it was not possible to\ndetermine whether symptomatic women may harbor viral particles in their oocytes.\nAdditionally, the test was performed only on oocytes, and the detection limit of the\nPCR test was 100 copies per ml in that study [ \n20 ]. It is hypothesized that even if the virus is not detectable in the\noocytes, it might have undesirable effects on the oocytes in other ways, such as\naltered metabolism, toxins, or changes within the ovarian follicle or ovarian niche\n[ 19 ]. In a study on postmenopausal women with\nsevere COVID-19, all patients were tested for SARS-CoV-2 in vaginal fluid, and all\nsamples were negative for the virus [ 28 ].\nWe agree with the suggested strategy that during the COVID-19 pandemic, even for\nfertility preservation in cancer patients, it is better to explore follicular fluid\nand seminal plasma for the virus before cryopreservation [ 29 ]. However, it is argued that ART procedures and repeated\nwashings might eliminate the viral load and ART embryo development, and the\nfollowing ART resulting in pregnancy might be even safer than a natural pregnancy\nduring the COVID-19 pandemic [ 30 ].\nThe present study had some limitations. First, the detection limit of the real-time\nPCR kit was 200 copies per ml, and lower viral loads might have been missed. Second,\nthe number of patients and samples was small.\n\nWe detected SARS-CoV-2 RNA in one follicular fluid sample out of\nfour. However, the other granulosa cells and oocyte samples were negative\nregarding viral RNA. Given the possible negative effects of the virus on human\nreproduction, the presence of SARS-CoV-2 RNA in the follicular fluid mandates\nstrict precautions to be taken with ART. Nevertheless, it is mandatory to\nperform more extensive studies to confirm the findings of this study and to\ndetermine the exact possible effects of SARS-CoV-2 on human reproduction and\nART outcomes by following the resulting pregnancies.\n\nThe authors declare that there are no conflicts of\ninterest.","source_license":"CC-BY-4.0","license_restricted":false}