Detection of SARS-CoV-2 in Follicular Fluid of COVID-19 Infected Women

Background: The coronavirus disease-2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is now spread all over the world. This study was designed to assess the possibility of SARS-CoV-2 presence in follicular fluid, which may have harmful effects on normal ovulation and fertility. Method(s): Five women who were candidates for Assisted Reproductive Technique (ART) and had a COVID-19 PCRpositive test on the day of oocyte retrieval participated in the study. SARS-CoV-2 tests were performed on the follicular fluid obtained from these women. Result(s): SARS-CoV-2 RNA was detected only in one follicular fluid sample, and other follicular fluid samples were negative. Conclusion(s): Because the COVID-19 effect on human reproduction is unknown, exact precautions should be taken during this pandemic, especially for women applying for ART.

Past COVID-19: The Impact on IVF Outcomes Based on Follicular Fluid Lipid Profile.

Follicular fluid is an important component of follicle growth and development. Negative effects of COVID-19 on follicular function are still open. The aim of this work was to study the features of the lipid profile of follicular fluid and evaluate the results of the in vitro fertilization (IVF) program in women after COVID-19 to identify biomarkers with prognostic potential. The study involved samples of follicular fluid collected from 237 women. Changes in the lipid composition of the follicular fluid of patients who underwent COVID-19 in mild and severe forms before entering the IVF program and women who did not have COVID-19 were studied by mass spectrometry. Several lipids were identified that significantly changed their level. On the basis of these findings, models were developed for predicting the threat of miscarriage in patients who had a severe course of COVID-19 and models for predicting the success of the IVF procedure, depending on the severity of COVID-19. Of practical interest is the possibility of using the developed predictive models in working with patients who have undergone COVID-19 before entering the IVF program. The results of the study suggest that the onset of pregnancy and its outcome after severe COVID-19 may be associated with changes in lipid metabolism in the follicular fluid.

Failure to Detect Viral Ribonucleic Acid in Follicular Fluid of a Severe Acute Respiratory Syndrome Coronavirus 2-Infected Female - A Report from the Indian Subcontinent.

Till today, we are still not clear about the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on reproductive tissues and its vertical transmission through the gametes and embryos. There are few case reports that aim to detect the presence or absence of SARS-CoV-2 viral ribonucleic acid (RNA) in the follicular fluid of the infected females. We hereby present the case report of a 33-year-old woman who was undergoing intracytoplasmic sperm injection with testicular sperm aspiration and was found to be positive for SARS-CoV-2 infection. The follicular fluid analysis failed to detect the presence of viral RNA in the sample. Ours is the first case report from India trying to detect the viral RNA from the follicular fluid of a female with SARS-CoV-2 positive status.

SARS-CoV-2 RNA in Follicular Fluid, Granulosa Cells, and Oocytes of COVID-19 Infected Women Applying for Assisted Reproductive Technology

Background: The Coronavirus disease 2019 (COVID-19) pandemic has raised concerns regarding the application of assisted reproductive technology (ART) in the world. Many ART programs have been delayed or continued with new precautions due to the ambiguity about vertical transmission and pregnancy safety. Regarding the possible risks of SARS-CoV-2 infection on ART and the resultant embryos, this study aimed to investigate the presence of SARS-CoV-2 in follicular fluid, granulosa cells, and oocytes of COVID-19-infected women undergoing ART. Materials and Methods: COVID-19-positive polymerase chain reaction tests were reported for five women undergoing ART cycles on the day of oocyte retrieval. SARS-CoV-2 tests were performed on oocytes, granulosa cells, and follicular fluid obtained from these COVID-19-infected women. Results: SARS-CoV-2 RNA was detected only in one follicular fluid sample;however, other follicular fluid samples, granulosa cells, and oocytes were negative regarding viral RNA. Conclusion: Given the unknown effects of COVID-19 on human reproduction and ART, strict precautions should be taken during the COVID-19 pandemic. [GMJ.2022;11:e2638] DOI:10.31661/gmj.v11i.2638

Are the Follicular Fluid Characteristics of Recovered Coronavirus Disease 2019 Patients Different From Those of Vaccinated Women Approaching in vitro Fertilization?

The aim of this pilot study is to evaluate if SARS-CoV-2 infection or vaccination against SARS-CoV-2 infection induce observable metabolic effects in follicular fluid of women who are following in vitro fertilization (IVF) treatments. The possible impact of coronavirus disease 2019 (COVID-19) on fertility and IVF outcome is considered. We have selected for this study: six women vaccinated against SARS-CoV-2 infection, five recovered COVID-19 patients, and we used nine healthy women as the control group. At the time of oocytes retrieval from participants in the study, follicular fluids were collected and metabolomic analysis was performed by 1H NMR spectroscopy in combination with multivariate analysis to interpret the spectral data. The search for antibody positivity in the follicular fluid aspirates was also carried out, together with the western blotting analysis of some inflammatory proteins, interleukin-6, tumor necrosis factor α (TNFα), and the free radical scavenger superoxide dismutase 2. Higher levels of Ala and Pro together with lower levels of lipids and trimethylamine N-oxide (TMAO) were found in follicular fluids (FFs) of vaccinated women while lower levels of many metabolites were detected in FFs of recovered COVID patients. Expression level of TNF-α was significantly lower both in recovered COVID-19 patients and vaccinated women in comparison to healthy controls.

Significant changes in follicular fluid phthalate metabolite levels reflect the lifestyle changes brought about by the strict COVID-19 lockdown in India.

OBJECTIVE: To assess if the unprecedented changes in lifestyle because of the lockdown initiated by the COVID-19 pandemic, which altered human behavior, and influenced purchase and consumption patterns, may have had an impact on the exposure to phthalates in Indian women undergoing in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). To evaluate if the effects of the strict and lengthy lockdown in India, which promoted the new norms of stay-at-home and work-from-home, closure of beauty parlors, and restriction on public gatherings, may have contributed to a decrease in the exposure to phthalates like dibutyl phthalate and diethyl phthalate. These chemicals are found in many personal care products (PCPs) which include cosmetics and fragrances. To investigate if the extensive use of flexible single-use plastic in personal protective equipment like face masks/gloves and in plastic packaging used for online purchases, food takeaways, and home deliveries of essentials and groceries during the COVID-19 pandemic, in an attempt to provide a contact-free delivery system may have inadvertently led to an increase in exposure to phthalates like di(2-ethylhexyl) phthalate, di-isononyl phthalate, and di-isodecyl phthalate which are plasticizers used in manufacturing flexible plastic. DESIGN: A comparative study of the levels of six phthalate metabolites detected in follicular fluid (FF) of Indian women undergoing IVF/ICSI 1 year before and immediately after the lockdown initiated by the COVID-19 pandemic. SETTING: In vitro fertilization center in a large referral hospital in India. PATIENT(S): A total of 176 Indian women seeking treatment for infertility and undergoing oocyte retrieval were included after obtaining consent. Each woman contributed one FF sample to the study. Group A (n = 96) women (mean age, 34.0 [±3.9] years, and mean BMI, 25.4 [±4.8]) had their FF samples collected and screened between January 2019 and mid-March 2020, 1 year before the lockdown. Group B (n = 80) women (mean age, 33.9 [±4.1] years, and mean BMI, 25.0 [±4.4]) had their FF collected and screened post the lockdown between October 2020 and June 2021. Both groups were matched by age and BMI. INTERVENTION(S): The cryopreserved FF samples of 176 women were processed using enzymatic deconjugation as well as the solid-phase extraction technique, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect the total levels of six phthalate metabolites. MAIN OUTCOME MEASURE(S): To evaluate the impact of the COVID-19 lockdown on the change in the phthalate metabolite levels in the FF of Indian women undergoing IVF/ICSI pre and post the lockdown. RESULT(S): The median levels of mono-n-butyl phthalate (1.64 ng/ml in group A vs. 0.93 ng/ml in group B; P<.001) and mono-ethyl phthalate (5.25 ng/ml in group A vs. 3.24 ng/ml in group B; P<.001) metabolites of dibutyl phthalate and diethyl phthalate found in PCPs including cosmetics and fragrances were significantly higher in the FF of 96 women (group A) compared with the levels seen in the FF of 80 women (group B). However, the median levels of mono-isononyl phthalate (0.11ng/ml in group A vs. 0.13 ng/ml in group B; P<.001), mono-isodecyl phthalate (0.11 ng/ml in group A vs. 0.14 ng/ml in group B; P<.001), and mono(2-ethyl-5-oxohexyl) phthalate (0.13 ng/ml in group A vs. 0.14 ng/ml in group B; P=.023) metabolites of di-isononyl phthalate, di-isodecyl phthalate, and di(2-ethylhexyl) phthalate used as plasticizers were significantly higher in the FF of women in group B compared with women in group A. CONCLUSION(S): The significant drop in mono-n-butyl phthalate and mono-ethyl phthalate levels, accumulated in the FF of 80 Indian women in group B reflects a decrease or absence of usage patterns of PCPs, including cosmetics and fragrances, thereby suggesting that these women may have deprioritized their use during the COVID-19 pandemic giving preference to personal hygiene and safety. Whereas the unprecedented increase in the use of flexible single-use plastic that became our first line of defense against the coronavirus during the COVID-19 pandemic might be responsible for the accumulation of significantly higher levels of mono-isononyl phthalate, mono-isodecyl phthalate, and mono(2-ethyl-5-oxohexyl) phthalate in FF of the same group.

Does mRNA SARS-CoV-2 vaccine in the follicular fluid impact follicle and oocyte performance in IVF treatments?

PROBLEM: The COVID-19 pandemic has many clinical manifestations. Rapid vaccine development raised concerns and speculations about future fertility outcomes and vaccine safety. We evaluated the effect of Pfizer-BioNTech mRNA SARS-CoV-2 vaccine on IVF treatment, oocyte and embryo quality, and pregnancy outcomes. METHOD OF STUDY: This prospective, observational cohort study was conducted in a referral IVF Unit, 3/2021-5/2021. We aimed to recruit all women undergoing IVF/ICSI cycles from 3/1-4/30/2021, 2-8 weeks after the second vaccination, and to analyze 50-60 samples in the 2-month period. Patients were categorized according to serum antibody levels: positive for spike (S), positive for nucleotide (N), or negative for both. On the day of ovum pick-up, follicular fluid and blood samples were analyzed for anti-nucleotide (anti-N) antibodies, and anti-spike (anti-S) antibodies, hormonal profile, C-reactive protein (CRP) and other metabolic parameters. RESULTS: Of 59 women enrolled, 37 reported being vaccinated and 22 were not. We found 97% correlation between anti-S and anti-N in the blood and the follicular fluid. Follicular fluid was analyzed based on antibody categorization. All IVF treatment parameters in the follicular fluids and serum were comparable, except CRP was significantly elevated among patients with anti-N antibodies (2.29 [1.42-6.08] vs. 4.11 [1.62-5.75] vs. 1.44 [.36-8.33]; p < .001). Pregnancy outcomes were comparable (44% vs. 33% vs. 50%; p = .97). CONCLUSION: mRNA SARS-CoV-2 vaccine did not appear to affect treatment outcomes or ovarian reserves in the subsequent IVF cycle.

The Effects of SARS-CoV-2 Infection on Female Fertility: A Review of the Literature.

As the coronavirus pandemic is far from ending, more questions regarding the female reproductive system, particularly fertility issues, arise. The purpose of this paper is to bring light upon the possible link between COVID-19 and women's reproductive health. This review emphasizes the effect of SARS-CoV-2 on the hormones, endometrium and menstrual cycle, ovarian reserve, follicular fluid, oocytes, and embryos. The results showed that endometrial samples did not express SARS-CoV-2 RNA. Regarding the menstrual cycle, there is a large range of alterations, but they were all reversible within the following months. The ovarian reserve was not significantly affected in patients recovering from both mild and severe infection in most cases, except one, where the levels of AMH were significantly lower and basal follicle-stimulating hormone (FSH) levels were increased. All COVID-19 recovered patients had positive levels of SARS-CoV-2 IgG in the follicular fluid. The amount of retrieved and mature oocytes and the fertilization rate were unharmed in three studies, except for one study, where the quantity of retrieved and mature oocytes was reduced in patients with higher levels of SARS-CoV-2 antibodies. The numbers of blastocysts, top-quality embryos, and euploid embryos were affected in most of the studies reviewed.

Undetectable viral RNA in follicular fluid, cumulus cells, and endometrial tissue samples in SARS-CoV-2-positive women.

OBJECTIVE: To study the presence of viral RNA in the follicular fluid, cumulus cells, and endometrial tissue samples in SARS-CoV-2-positive women undergoing assisted reproductive technology (ART). DESIGN: Prospective, single-center, observational study. SETTING: Tertiary hospital. PATIENT(S): A total of 16 patients undergoing transvaginal oocyte retrieval who had a positive SARS-CoV-2 RNA test <48 hours before the procedure. All patients underwent the retrieval between September 2020 and June 2021 and used in vitro fertilization or intracytoplasmic sperm injection. All embryos were vitrified to avoid conception during SARS-CoV-2 infection. INTERVENTION(S): Follicular fluid aspirated during oocyte retrieval, cumulus cells, and endometrial samples were analyzed for SARS-CoV-2 RNA using the RealStar SARS-CoV-2 RT-PCR-Kit1.0. MAIN OUTCOME MEASURE(S): The primary outcome parameter was the detection of viral RNA in the follicular fluid, cumulus cells, and endometrial cells. Fertilization rate, embryo developmental potential, and clinical outcome after frozen embryo transfer were secondary outcome parameters. RESULT(S): Samples from 16 patients were analyzed. Cycle threshold values of <40 were considered positive. All samples were negative for SARS-CoV-2 viral RNA. No inflammatory lesions of the endometrium were identified histologically. Fertilization rate, embryo development, and clinical outcomes after embryo transfer were reassuring. CONCLUSION(S): In women infected with SARS-CoV-2 who underwent ART, viral RNA was undetectable in the follicular fluid, cumulus cells, and endometrium. Caution is warranted in view of the small sample size, and the risk of SARS-CoV-2 affecting the embryo via ART cannot be ruled out. Adequate counseling of women and couples undergoing ART is crucial in parallel with further research on the effect of exposure of the early human embryo to SARS-CoV-2. CLINICAL TRIAL REGISTRATION NUMBER: NCT04425317.

Risk of contamination of semen, vaginal secretions, follicular fluid and ovarian medulla with SARS-CoV-2 in patients undergoing ART.

STUDY QUESTION: Can severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA be detected in the reproductive tract of asymptomatic patients undergoing ART? SUMMARY ANSWER: SARS-CoV-2 mRNA is not detectable in semen, follicular fluid, vaginal secretions or residual medulla from ovarian tissue cryopreservation procedures in asymptomatic patients who undergo ART, irrespective of the results of a triage questionnaire and a nasopharyngeal SARS-CoV-2 RNA detection test. WHAT IS KNOWN ALREADY: The SARS-CoV-2 pandemic had a huge impact on the activities of fertility clinics. Although some studies reported the presence of SARS-CoV-2 mRNA in the reproductive system during or after acute COVID-19 symptomatic infections, uncertainties remain regarding the presence of viral mRNA in the reproductive material and follicular fluid of asymptomatic patients undergoing ART. STUDY DESIGN, SIZE, DURATION: An observational cohort trial of residual material samples including semen, follicular fluid, vaginal secretions and ovarian medulla was conducted during the second pandemic wave in Brussels from September 2020 to April 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients who underwent ART (IUI, IVF/ICSI, oocyte and ovarian tissue cryopreservation) responded to a triage questionnaire at the beginning and end of the cycle and underwent nasopharyngeal swab collection for SARS-CoV-2 RNA detection by RT-PCR before the procedure according to standard recommendations. For semen analysis, only the questionnaire was requested the day before the sample collection. The ART cycles of patients with positive nasopharyngeal SARS-CoV-2 RNA detection tests and/or questionnaires were cancelled except for those that could not be postponed. After providing informed consent, swabs on residual materials were collected the day of the oocyte, ovarian tissue or semen collection and were processed for RT-qPCR. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 394 samples from 291 patients were analysed. Amongst them, 20 samples were obtained from patients with a positive questionnaire but negative nasopharyngeal SARS-CoV-2 test and 20 others were from patients with a positive nasopharyngeal SARS-CoV-2 test. The remaining samples were collected from patients with a negative or unknown nasopharyngeal SARS-CoV-2 test and/or a negative or unknown triage questionnaire. Viral RNA for SARS-CoV-2 was undetectable in all of the samples. LIMITATIONS, REASONS FOR CAUTION: Considering the cancellation policy, only a limited number of samples from patients with positive triage questionnaires or nasopharyngeal SARS-CoV-2 tests were included in the analysis. WIDER IMPLICATIONS OF THE FINDINGS: The study suggested that there was no risk of reproductive tract contamination by SARS-CoV-2 in asymptomatic patients, irrespective of the results from a triage questionnaire or nasopharyngeal SARS-CoV-2 test. The results suggested that no additional measures to prevent staff or cross-patient contamination need to be implemented in the IVF and andrology laboratories. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Université Libre de Bruxelles and by a grant from Ferring. A.D. and I.D. received a grant from Ferring for the study. The authors have no other conflict of interest to declare related to this study. TRIAL REGISTRATION NUMBER: N/A.

SARS-CoV-2 infection negatively affects ovarian function in ART patients.

Several organs, such as the heart, breasts, intestine, testes, and ovaries, have been reported to be target tissues of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To date, no studies have demonstrated SARS-CoV-2 infection in the female reproductive system. In the present study, we investigated the effects of SARS-CoV-2 infection on ovarian function by comparing follicular fluid (FF) from control and recovered coronavirus disease 2019 (COVID-19) patients and by evaluating the influence of these FF on human endothelial and non-luteinized granulosa cell cultures. Our results showed that most FFs (91.3%) from screened post COVID-19 patients were positive for IgG antibodies against SARS-CoV-2. Additionally, patients with higher levels of IgG against SARS-CoV-2 had lower numbers of retrieved oocytes. While VEGF and IL-1ß were significantly lower in post COVID-19 FF, IL-10 did not differ from that in control FF. Moreover, in COV434 cells stimulated with FF from post COVID-19 patients, steroidogenic acute regulatory protein (StAR), estrogen-receptor ß (Erß), and vascular endothelial growth factor (VEGF) expression were significantly decreased, whereas estrogen-receptor α (ERα) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) did not change. In endothelial cells stimulated with post COVID-19 FF, we observed a decrease in cell migration without changes in protein expression of certain angiogenic factors. Both cell types showed a significantly higher γH2AX expression when exposed to post COVID-19 FF. In conclusion, our results describe for the first time that the SARS-CoV-2 infection adversely affects the follicular microenvironment, thus dysregulating ovarian function.

Failure to Detect Viral RNA in Follicular Fluid Aspirates from a SARS-CoV-2-Positive Woman.

Although there is no known difference between the clinical manifestations of SARS-CoV-2 in pregnant and non-pregnant women based on the studies published until now, in vitro fertilization (IVF) treatments were suspended during the pandemic due to uncertainties with the suggestions of associated societies. However, we do not have enough data on the exact effects of SARS-CoV-2 on fertility and pregnancy and whether there are damaging effects on IVF outcome. There is no available evidence about the transmission of SARS-CoV-2 by either sexual way or through intrauterine insemination (IUI) or IVF. Up until now, there is no report to document the presence or absence of viral RNA in follicular fluid of SARS-CoV-2-positive women. In this paper, we present a case of oocyte retrieval from a SARS-CoV-2-positive woman and the search for viral RNA by polymerase chain reaction (PCR) in the follicular fluid aspirates.

Validation of the French IVF guidelines during the COVID-19 pandemic.

RESEARCH QUESTION: Is a symptom questionnaire as per the French IVF guidelines adequate for screening patients during the COVID-19 pandemic? DESIGN: Patients planning IVF from June 2020 to February 2021 were included in the study. In compliance with French IVF guidelines, all patients fever-free on the day of oocyte retrieval were screened for risk of COVID-19 by completing a symptom questionnaire after being counselled regarding the importance of a COVID-19-free medical practice. Patients with IVF planned between June and September 2020 only completed the questionnaire (group 1), while those planning IVF after September 2020 also underwent the RT-PCR test for SARS-CoV-2 RNA (group 2). Cycle cancellation rates between groups were compared. Group 1 patients consented for follicular fluid testing for SARS-CoV-2 and an interview after cycle completion to determine COVID-19 exposure during the 6 months before and after retrieval. RESULTS: Cycle cancellation rates for groups 1 and 2 were 0% (0/214) versus 1.4% (8/577), respectively, (P = 0.116). All 183 follicular fluid samples from group 1 were negative for SARS-CoV-2 RNA. Of 171 patients interviewed post-IVF, 16 (93.4%) developed COVID-19 symptoms or a positive real-time PCR (RT-PCR) RT-PCR test, but none within 2 months pre- or post-retrieval. CONCLUSIONS: These results provide reassurance that, consistent with the COVID-19 French IVF guidelines, use of a symptom questionnaire is effective in screening patients planning to undergo IVF. Failure to detect viral RNA in any follicular fluid sample does not negate the possibility that follicular fluid is a viral reservoir. However, the findings provide reassurance that the follicular environment in this study's carefully screened population was COVID-free.

Absence of SARS-CoV-2 (COVID-19 virus) within the IVF laboratory using strict patient screening and safety criteria.

RESEARCH QUESTION: Is there a risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral exposure and potential cross-contamination from follicular fluid, culture media and vitrification solution within the IVF laboratory using strict patient screening and safety measures? DESIGN: This was a prospective clinical study. All women undergoing transvaginal oocyte retrieval were required to have a negative SARS-CoV-2 RNA test 3-5 days prior to the procedure. Male partners were not tested. All cases used intracytoplasmic sperm injection (ICSI). The first tube of follicular fluid aspirated during oocyte retrieval, drops of media following removal of the embryos on day 5, and vitrification solution after blastocyst cryopreservation were analysed for SARS-CoV-2 RNA. RESULTS: In total, medium from 61 patients, vitrification solution from 200 patients and follicular fluid from 300 patients was analysed. All samples were negative for SARS-CoV-2 viral RNA. CONCLUSIONS: With stringent safety protocols in place, including testing of women and symptom-based screening of men, the presence of SARS-CoV-2 RNA was not detected in follicular fluid, medium or vitrification solution. This work demonstrates the possibility of implementing a rapid laboratory screening assay for SARS-CoV-2 and has implications for safe laboratory operations, including cryostorage recommendations.