Alexandra Alvergne
email: alexandra.alvergne@umontpellier.fr

Gabriella Kountourides

M Austin Argentieri

Lisa Agyen

Natalie Rogers

Dawn Knight

Gemma C Sharp

Jacqueline A Maybin

Zuzanna Olszewska

A retrospective case-control study on menstrual cycle changes following COVID-19 vaccination and disease

There has been increasing public concern that COVID-19 vaccination causes menstrual disturbance regarding the relative effect of vaccination compared to SARS-CoV-2 infection. Our objectives were to test potential risk factors for reporting menstrual cycle changes following COVID-19 vaccination and to compare menstrual parameters following COVID-19 vaccination and COVID-19 disease. We performed a secondary analysis of a retrospective online survey conducted in the UK in March 2021. In premenopausal vaccinated participants (n=4,989), 18% reported menstrual cycle changes after their first COVID-19 vaccine injection. The prevalence of reporting any menstrual changes was higher for women who smoke, have a history of COVID-19 disease, or are not using oestradiol-containing contraceptives.

In a second sample including both vaccinated and unvaccinated participants (n=12,579), COVID-19 vaccination alone was not associated with abnormal menstrual cycle parameters while a history of COVID-19 disease was associated with an increased risk of reporting heavier bleeding, 'missed' periods and inter-menstrual bleeding.

Introduction

There has been substantial public concern that the COVID-19 pandemic has caused disruption of menstrual cycles due to vaccination, [START_REF] Mcshane | Can the vaccine make your period worse? These women say yes[END_REF][START_REF] Saar | Women Say COVID Vaccine Side Effects Impact Their Periods, So Why Don't Doctors Care? Haaretz[END_REF][START_REF] Efrati | Some Vaccinated Israeli Women Report Irregular Menstrual Cycles[END_REF] infection with the SARS-CoV-2 virus [START_REF] Lomte | Impact of SARS-CoV-2 infection on menstrual cycle[END_REF] , pandemic-related stress and lifestyle changes. [START_REF] Morgan | Pandemic Periods: WhyWomen's Menstrual Cycles Have Gone Haywire[END_REF] Yet, the independent contribution of each factor to menstrual cycle changes remains understudied, [START_REF] Sharp | The COVID-19 pandemic and the menstrual cycle: research gaps and opportunities[END_REF][START_REF] Wang | A prospective study of the association between SARS-CoV-2 infection and COVID-19 vaccination with changes in usual menstrual cycle characteristics[END_REF] particularly prior to media attention to the topic. This is despite rising awareness among clinicians that the menstrual cycle should be used as a vital sign of female health [START_REF]Menstruation in Girls and Adolescents: Using the Menstrual Cycle as a Vital Sign (2015)[END_REF][START_REF] Li | Characterizing physiological and symptomatic variation in menstrual cycles using self-tracked mobile-health data[END_REF] and that sex is a biological variable which should be considered in immunological studies. [START_REF] Klein | Sex differences in immune responses[END_REF] Ultimately, the lack of data for investigating independent associations between menstrual cycles and both COVID-19 vaccines and SARS-CoV-2 infection limit our ability to clarify the impact of the COVID-19 pandemic on menstruation. [START_REF] Carp-Veliscu | The Effects of SARS-CoV-2 Infection on J o u r n a l P r e -p r o o f Female Fertility: A Review of the Literature[END_REF] Such knowledge is critical for advising women about the relative risk of experiencing menstrual disturbance when getting vaccinated against COVID-19 versus infected with SARS-CoV-2.

Before the COVID-19 pandemic, research on the relationship between vaccination and menstrual cycle health had been limited to the prophylactic typhoid [START_REF] Lamb | Experiences with prophylactic Typhoid vaccination[END_REF] , HPV [START_REF] Suzuki | No association between HPV vaccine and reported post-vaccination symptoms in Japanese young women: Results of the Nagoya study[END_REF][START_REF] Gong | Human papillomavirus vaccine-associated premature ovarian insufficiency and related adverse events: data mining of Vaccine Adverse Event Reporting System[END_REF] and hepatitis B vaccines [START_REF] Shingu | Menstrual Abnormalities after Hepatitis B Vaccine[END_REF] . However, recent reports of menstrual disturbances following COVID-19 vaccination in the media [START_REF] Mcshane | Can the vaccine make your period worse? These women say yes[END_REF][START_REF] Saar | Women Say COVID Vaccine Side Effects Impact Their Periods, So Why Don't Doctors Care? Haaretz[END_REF][START_REF] Efrati | Some Vaccinated Israeli Women Report Irregular Menstrual Cycles[END_REF] and surveillance schemes (e.g., in the UK [START_REF] Male | Menstrual changes after covid-19 vaccination[END_REF][START_REF] Andrew | 4,000 women report period problems after Covid jab[END_REF] and France [START_REF]Vaccin contre le Covid-19 : les femmes qui subissent des troubles menstruels sont invitées à les déclarer[END_REF] ) have led to a surge of research. [START_REF] Wang | A prospective study of the association between SARS-CoV-2 infection and COVID-19 vaccination with changes in usual menstrual cycle characteristics[END_REF][START_REF] Rodríguez Quejada | Menstrual cycle disturbances after COVID-19 vaccination[END_REF][START_REF] Alvergne | Effect of COVID-19 vaccination on the timing and flow of menstrual periods in two cohorts[END_REF][START_REF] Muhaidat | Menstrual Symptoms After COVID-19 Vaccine: A Cross-Sectional Investigation in the MENA Region[END_REF][START_REF] Lee | Investigating trends in those who experience menstrual bleeding J o u r n a l P r e -p r o o f changes after SARS-CoV-2 vaccination[END_REF][START_REF] Trogstad | Increased Occurrence of Menstrual Disturbances in 18-to 30-Year-Old Women after COVID-19 Vaccination[END_REF] Prospective studies using samples of app users not using hormonal contraception found that COVID-19 vaccination changed cycle length by < 1 day, [START_REF] Edelman | Association Between Menstrual Cycle Length and Coronavirus Disease 2019 (COVID-19) Vaccination[END_REF][START_REF] Edelman | Association between menstrual cycle length and covid-19 vaccination: global, retrospective cohort study of prospectively collected data[END_REF][START_REF] Gibson | Covid-19 vaccination and menstrual cycle length in the Apple Women's Health Study[END_REF] with similar findings in a prospective study of 3,858 pre-menopausal health professionals. [START_REF] Wang | A prospective study of the association between SARS-CoV-2 infection and COVID-19 vaccination with changes in usual menstrual cycle characteristics[END_REF] In a recent prospective study of 79 J o u r n a l P r e -p r o o f participants recruited via social media, the subsequent menstrual episode following COVID-19 vaccination occurred a mean of 2.3 days late after dose 1 and 1.3 days late after dose 2. [START_REF] Alvergne | Effect of COVID-19 vaccination on the timing and flow of menstrual periods in two cohorts[END_REF] Beyond cycle length, other studies have reported various changes in regularity, duration and volume. [START_REF] Rodríguez Quejada | Menstrual cycle disturbances after COVID-19 vaccination[END_REF][START_REF] Alvergne | Effect of COVID-19 vaccination on the timing and flow of menstrual periods in two cohorts[END_REF] For instance, in a sample of young participants (18-30 years) drawn at random from the Norwegian National Population Registry, heavy bleeding increased from 7.6% to 13.6% in the first cycle after vaccination, and from 8.2% to 15.3% after the second vaccine dose [START_REF] Trogstad | Increased Occurrence of Menstrual Disturbances in 18-to 30-Year-Old Women after COVID-19 Vaccination[END_REF] . Recent data from a genderdiverse sample receiving COVID-19 vaccination in the US suggests that changes in the form of heavy and breakthrough bleeding affect many people. [START_REF] Lee | Investigating trends in those who experience menstrual bleeding J o u r n a l P r e -p r o o f changes after SARS-CoV-2 vaccination[END_REF] While there is accumulating evidence that COVID-19 vaccination-related menstrual symptoms are associated with small and temporary changes in cycle length [START_REF] Rodríguez Quejada | Menstrual cycle disturbances after COVID-19 vaccination[END_REF][START_REF] Edelman | Association Between Menstrual Cycle Length and Coronavirus Disease 2019 (COVID-19) Vaccination[END_REF] , there has been no quantitative assessment of the risk factors for menstrual disturbances following COVID-19 vaccination prior to widespread media attention.

Contrasting with the emerging picture showing a small effect of COVID-19 vaccine on cycle length, research on the associations between SARS-CoV-2 infection and menstrual cycle changes is scarce and inconsistent. [START_REF] Carp-Veliscu | The Effects of SARS-CoV-2 Infection on J o u r n a l P r e -p r o o f Female Fertility: A Review of the Literature[END_REF][START_REF] Lebar | The Effect of COVID-19 on the Menstrual Cycle: A Systematic Review[END_REF] Early in the pandemic, a cross-sectional hospital-based study conducted in China and including COVID-19 patients admitted to hospital (n=177) and controls (n=91), found that COVID-19 patients reported more changes in menstrual blood volume (control versus COVID-19, 5% versus 25%, P < 0.001) and cycle length (control versus COVID-19, 6% versus 28%, P < 0.001). [START_REF] Li | Analysis of sex hormones and menstruation in COVID-19 women of child-bearing age[END_REF] Note that the external validity of this study has been questioned as the sample is biased towards women with multisystem dysfunction. [START_REF] Danesh | The effects of SARS-CoV-2 on menstruation[END_REF] In a sub-sample of 127 participants aged 18-45 years taken from a prospective cohort study of SARS-

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CoV-2 positive cases (Arizona CoVHORT study), 16% reported changes in their menstrual cycle, including irregular menstruation (60%), increase in premenstrual symptoms (45%) and infrequent menstruation (35%). [START_REF] Khan | SARS-CoV-2 infection and subsequent changes in the menstrual cycle among participants in the Arizona CoVHORT study[END_REF] Yet causality cannot be inferred in this study due to the absence of a control group. Conversely, an association between SARS-CoV-2 infection and cycle changes was not observed in a prospective study of 3,858 pre-menopausal health professionals taking part in the Nurses' Health Study 3. [START_REF] Wang | A prospective study of the association between SARS-CoV-2 infection and COVID-19 vaccination with changes in usual menstrual cycle characteristics[END_REF] In this sample, the prevalence of infection was low (n=421, 11%) compared to vaccination (n=3,527, 91%) and more than half of COVID positive individuals (n=223) were vaccinated prior to infection, [START_REF] Wang | A prospective study of the association between SARS-CoV-2 infection and COVID-19 vaccination with changes in usual menstrual cycle characteristics[END_REF] which may have limited the ability of the study to detect small to moderate effects. Finally, in a study of 187 American women, having detectable SARS-CoV-2 IgG antibodies was associated with a higher percentage of self-reported menstrual irregularities (cycles not between 26-35 days in the 3 months prior to survey) among unvaccinated women, [START_REF] Cherenack | Infection with SARS-CoV-2 is associated with menstrual irregularities among women of reproductive age[END_REF] suggesting that SARS-CoV-2 may lead to abnormal cycle parameters. A study better powered to evaluate the independent association of SARS-CoV-2 and abnormal cycle changes is needed to inform vaccination decisions.

Objectives of the study

The objectives of this study were three-fold: (1) to identify the risk factors for reporting any menstrual changes following COVID-19 vaccination, (2) to evaluate the independent effect of COVID-19 disease and COVID-19 vaccination on menstrual parameters as defined by the International Federation of Gynaecologists and Obstetricians (FIGO), [START_REF] Munro | The two FIGO systems for normal and abnormal uterine bleeding symptoms and classification of causes of abnormal uterine bleeding in the reproductive years: 2018 revisions[END_REF] including menstrual frequency, regularity, duration, volume and inter-menstrual bleeding, (3) to capture the types and breadth of menstrual J o u r n a l P r e -p r o o f disturbances following COVID-19 vaccination in participants' written accounts. To do this, we used a large retrospective cross-sectional study on menstruation somewhat representative of those who menstruate in the UK. This was launched before UK media coverage of concerns over menstrual vaccine side-effects and includes both quantitative and textual data on menstrual cycle changes.

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Results

Self-reported menstrual cycle changes following COVID-19 vaccination

Sample characteristics

Out of the 26,710 individuals who completed the survey, 8,539 (31%) reported having been vaccinated, with either one (n=7,270) or two doses (n=1,269). Although the UK vaccination campaign began by targeting older and at-risk populations, we did not observe an over-representation of those over 40 years old. Of note, 54% of participants were nulliparous and 49% had a university or college degree. We excluded participants who did not have a period in the 12 months preceding the survey, those who were post-menopausal or transitioning, breastfeeding or pregnant, and among those who selected "Other changes", those who contributed text to the effect of "too early to say" when describing menstrual disturbances following COVID-19 vaccination (n=369, 64% of those selecting the answer "Other changes") (Figure 1). The final sample size of vaccinated individuals was 4,989, of which 53% received the Oxford-AstraZeneca and 47% the Pfizer BioNTech vaccine (Table 1). The median age was 35 years old (IQR: 28 to 43), with most participants living in England (81%), self-reporting as white (95%) and self-identifying as women (99%).

Risk factors for COVID-19 vaccine-related changes in menstrual cycles

Eighty-two percent of eligible participants reported no changes to their menstrual cycles following COVID-19 vaccination. Only 6.2% reported more disruption, 1.6% reported less disruption and 10.2% reported "Other changes", which could be J o u r n a l P r e -p r o o f interpreted as any changes in cycle length and regularity, period duration and volume of menstrual bleeding as well as premenstrual symptoms.

The univariable analyses show that reporting any changes to menstrual cycles after COVID-19 vaccination is associated with contraceptive type, smoking behaviour, COVID-19 disease history and menstrual cycle changes over the last year (Figure 2).

Reporting changes to menstrual cycles after COVID-19 vaccination was not associated with age, body mass index, ethnic group, gender, marital status, physical activity, income, education, place of residence, cycle length, period length, irregular cycles, heavy bleeding, vaccine type, vaccine timing, parity, life satisfaction changes, medication use, use of vitamins/supplements, endometriosis, polycystic ovary syndrome, thyroid disease, uterine polyps, uterine fibroids, inter cystitis and eating disorders (Figure 2; Table S1).

The multivariable analyses show that the prevalence of menstrual cycle changes after COVID-19 vaccination is 33% lower among users of combined contraceptives S1). The effects remain significant after adjusting for self-J o u r n a l P r e -p r o o f reported overall magnitude of menstrual cycle changes over the year preceding the survey which is positively associated with the risk of reporting any changes (PR=1.13, 95CI=[1.05 to 1.21], P=0.003). The findings were replicated when using complete case analyses with unimputed data, indicating that the results are not an artefact of the missing data imputation process (Table S2).

Risk for 'abnormal' menstrual characteristics

Sample characteristics

To investigate independent effects of COVID-19 vaccination and COVID-19 disease on abnormal menstrual parameters as defined by the FIGO criteria for Abnormal Uterine Bleeding [START_REF] Munro | The two FIGO systems for normal and abnormal uterine bleeding symptoms and classification of causes of abnormal uterine bleeding in the reproductive years: 2018 revisions[END_REF] , we conducted additional analyses including participants who were not vaccinated, leading to a final sample of 12,579 (Figure 4). We compared menstrual cycle parameters across 4 groups (Table 2) S3).

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Cycle parameters

Cycle frequency For this analysis we excluded participants who reported "Too irregular to say" for the outcome variable "Cycle length during the pandemic" (n=889), as we were interested in ascribing frequency. Across all groups of remaining participants (n=11,690), the most probable outcome is to report normal cycles (between 24 and 38 days, 70.2%), followed by frequent (<24 days, 26.4%) and infrequent cycles (>38 days, 3.3%, Figure 5). The relative risk of frequent vs. normal cycles and the relative risk of infrequent vs. normal cycles do not vary significantly between the vaccinated only group and the control group (no vaccination and no infection), suggesting vaccination alone does not associate with abnormal cycle frequency (Table S3, Figure 5). However, compared to being vaccinated only, a history of COVID-19 disease increases the relative risk of frequent vs. normal cycles by 30% Finally, the odds for reporting "missed" and/or "stopped" periods do not vary between the control group and the vaccinated-only group (Control: PR = 0.96, 95CI = [0.82 to 1.13], FDR P-value = 0.62), but increase by 27% in the COVID-19 disease-only group (Cov: PR = 1.27, 95CI = [1.05 to 1.54], FDR P-value = 0.032, Table S3), with the J o u r n a l P r e -p r o o f probability of reporting missing or stopped periods increasing from 7% in the vaccinated-only group to 9% in the COVID-19 disease-only group (Figure 5). A significant increase is not observed for participants who are also both infected and vaccinated (Covax: PR = 1.14, 95CI = [0.92 to 1.41], FDR P-value = 0.296). Baseline cycle frequency and contraceptive and reproductive disease at baseline do not influence the association between a history of COVID-19 and cycle frequency during the pandemic (models including interaction effects are worse fits to the data than a model without interaction, Table S4).

Cycle regularity Across all groups of participants, the most probable outcome is to report regular cycles at the time of survey (less than 10 days difference between shortest and longest cycles, 79.7%), followed by highly irregular (over 20 days difference, 10.5%) and somewhat irregular (between 10-20 days difference, 9.8%, Figure 5). The relative risks of reporting irregular vs. regular cycles are not associated with COVID-19 vaccination and disease history in this sample (Table S3, Figure 5).

Period duration

There are no significant differences in the prevalence of periods longer than 8 days between the vaccinated-only group and the control group (PR = 1.05, 95CI [0.74; 1.49], FDR P-value = 0.8284, Table S3, Figure 5). Compared to the vaccinated-only group, the prevalence of periods longer than 8 days is increased by 65% for the group combining both COVID-19 vaccination and disease (PR = 1.65, 95CI [1.08; 2.54], FDR P-value = 0.0474), a tendency not observed for those with a history of COVID-19 disease only (PR = 1.44, 95CI [0.94; 2.21], FDR P-value = 0.1446, Table S3). The associations do not depend on initial period length category, reproductive J o u r n a l P r e -p r o o f disease at baseline or contraceptive uptake in this dataset as models including an interaction between any of those variables and COVID-19 vaccination and disease history are worse fits to the data than a model without interaction (Table S4).

Flow volume Across all groups of participants, the most probable outcome is 'No changes' (40.9%), followed by 'heavier' (25.1%), 'heavier and lighter' (19.1%) and 'lighter' (14.9%). There are no significant differences between the vaccinated-only and the control groups for the relative risks of 'heavier' vs. 'normal' periods (RRR = 0.96, 95CI = [0.85 to 1.1], FDR P-value = 0.752), 'lighter' vs. 'normal' periods or 'lighter and heavier' vs. 'normal' periods. As compared to being vaccinated only, a history of COVID-19 disease increases the risk of heavier vs. normal periods by ca. 38% (Cov: RRR = 1.38, 95CI = [1.17 to 1.63], FDR P-value = 0.0006; Covax: RRR = 1.39, 95CI = [1.16 to 1.66], FDR P-value = 0.0015) and the risk of 'lighter' periods vs. 'no changes' by 29% (Covax: RRR = 1.29, 95CI = [1.05 to 1.59], FDR P-value = 0.05). In absolute terms, the predicted probability of reporting heavier periods increases from 25% in the vaccinated-only group to 34% for participants in the COVID-19 only group (Figure 5).

The associations do not depend on initial period flow, reproductive disease at baseline or contraceptive uptake in this dataset as models including an interaction between any of those variables and COVID-19 vaccination and disease history are worse fits to the data than a model without interaction (Table S4).

Intermenstrual bleeding (IMB)

Across all groups of participants, the most probable outcome for spotting mid-cycle during the pandemic compared to before is 'no changes' (73%) followed by 'more' (18.5%), 'less' (3.1%) and 'sometimes more and J o u r n a l P r e -p r o o f sometimes less' (5.4%). There are no significant differences between the vaccinatedonly and the control groups for the relative risks of 'more' vs. 'no changes' for IMB (RRR = 0.99, 95CI = [0.85 to 1.15], FDR P-value = 0.953). As compared to the vaccinated-only group, the risk of reporting subjectively more spotting mid-cycle than pre-pandemic increases from 18% to 23% for participants with a history of COVID-19 disease (Cov: RRR = 1.31, 95CI [1.09; 1.58], P = 0.0149; Covax: RRR = 1.30, 95CI [1.06; 1.59], FDR P-value = 0.0338). The associations do not depend on reproductive disease at baseline or contraceptive uptake in this dataset as models including an interaction between any of those variables and COVID-19 vaccination and disease history are worse fits to the data than a model without interaction (Table S4). The findings remaining significant after Bonferroni correction (heavy bleeding and IMB) were replicated when using complete case analyses with unimputed data (Table S5).

Textual description of menstrual cycle changes following COVID-19 vaccination

Most common changes reported

The analysis of text written by participants who selected "Other changes" (n= 574, 57% of those reporting any changes) rather than "MORE disruption" or "LESS disruption" showed concerns over cycle length and menstrual bleeding patterns. The most common unigrams (individual words) were "late", "bleed", "early", "long", "heavy", "spotting", "short", "pain" and "stop" and the most common bigrams (pairs of adjacent words) were "day late", "period start", "heavy bleed", and "late period" (Figure 6). While many reported menstrual cycle changes that entailed heavier bleeding/periods, there J o u r n a l P r e -p r o o f was no one single pattern of symptoms, with changes including both early and late periods, and diverse experiences reported (from "miss period" to "heavy bleed").

Associations between symptoms

Only a few symptoms are correlated (φ < -0.2 or φ > 0.2). "Cramps" positively correlate with "pain" and "heavy" and "bleed" negatively correlates with "late". Further, "lighter" positively correlates with "normal", as participants report that "period was two days late, and lighter than normal". However, "lighter" and "late" do not co-occur more than expected by chance (Figure 7).

Clusters of words

Different clusters of symptoms emerge from the text, such as irregular periods, heavy cramps, and pain. However, the "pain" cluster encompassed many words that are weakly correlated, suggesting a diversity of pain experience. There was also some uncertainty regarding which changes do occur, with participants finding it "hard to say if the irregular periods are still due to covid or the vaccination". When only correlations >0.20 were considered (Figure 8), 4 clusters emerged: "heavy, painful, cramps", "irregular, disruption", "lot, clot", and an experiential cluster "symptom, experience, pain, increase, feel". Notably, various pain experiences that do not directly relate to menstrual cramps were reported in the main text, including stomach pain and headache.

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Meaning of the study

Most menstruating people in our sample (82%) did not experience menstrual changes following COVID-19 vaccination. Further, we did not find vaccination to be associated J o u r n a l P r e -p r o o f with "abnormal" cycle parameters, as defined by FIGO, and we found no difference in the risk of reporting frequent or infrequent cycles, irregular cycles, long period duration (+8 days), heavy periods or inter-menstrual bleeding between vaccinated-only participants and the control group (not vaccinated and without a history of COVID-19 disease). This provides reassuring data suggesting that COVID-19 vaccination will not lead to menstrual changes in most people, which can be helpful when counselling reproductive-aged women about COVID-19 vaccination and menstrual changes.

However, 18% did report menstrual disturbance following COVID-19 vaccination, a proportion that is above the threshold for a 'very common' (≥ 1/10) adverse reaction according to international pharmacovigilance standards. [START_REF] Neubert | E-pharmacovigilance: development and implementation of a computable knowledge base to identify adverse drug reactions[END_REF] For instance, the rate of menstrual cycle changes assessed through self-report is more frequent than systemic side-effects after the first dose of the Pfizer vaccine (13.5%), according to data collected in the COVID Symptom Study app. [START_REF] Menni | Vaccine side-effects and SARS-CoV-2 infection after vaccination in users of the COVID Symptom Study app in the UK: a prospective observational study[END_REF] Given the retrospective nature of the survey, we cannot attribute changes to the vaccine as participants may have perceived normal menstrual variability. Nevertheless, clinicians should consider counselling women about possible menstrual effects following COVID-19 vaccination, while emphasising the need to seek medical advice if they are severe and lasting more than one cycle or involving "red flag" symptoms such as inter-menstrual bleeding, post-coital bleeding, or post-menopausal bleeding. This study also suggests that current smoking and having had COVID-19 increase the risk of experiencing menstrual disturbance following COVID-19 vaccination and that those on the combined oral contraceptive pill (COCP) are less likely to experience menstrual disturbance. Knowledge of risk factors may help tailor advice to individuals who menstruate prior to COVID-19 vaccination.

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Risk factors for menstrual cycle changes following COVID-19 vaccination

Our finding that using combined oral contraceptives decreases the risk of reporting menstrual changes post-vaccination by 50% contrasts with those obtained by similar online surveys in the US [START_REF] Lee | Characterizing menstrual bleeding changes occurring after SARS-CoV-2 vaccination[END_REF] and in the UK. [START_REF] Male | Effect of COVID-19 vaccination on menstrual periods in a retrospectively recruited cohort[END_REF] While a previous US study found "very little difference between respondents with spontaneous and hormonally contracepting cycles in the rate of post-vaccine heavy menstrual flow", [START_REF] Lee | Characterizing menstrual bleeding changes occurring after SARS-CoV-2 vaccination[END_REF] a UK-based study found that "people on hormonal contraception were more likely to report a change to menstrual flow". [START_REF] Male | Effect of COVID-19 vaccination on menstrual periods in a retrospectively recruited cohort[END_REF] The authors of the latter study attribute their finding to a reporting bias, where people using hormonal contraception to decrease their blood flow may be particularly motivated to respond to the survey. [START_REF] Male | Effect of COVID-19 vaccination on menstrual periods in a retrospectively recruited cohort[END_REF] Of note is that the effect of hormonal contraception is not directly comparable across samples, as our study distinguished between oestradiol-containing and progestogen-only contraceptives, noting a decreased risk of reporting any menstrual changes only for those using oestradiolbased contraceptives. The protective effect of combined contraceptives for cycle changes post-vaccination has been replicated in another study. [START_REF] Alvergne | Effect of COVID-19 vaccination on the timing and flow of menstrual periods in two cohorts[END_REF] We found that smokers were more at risk of reporting menstrual disturbances following vaccination against COVID-19. Previous studies found that heavy smoking (> 20 cigarettes/day) was associated with a shortening of the follicular phase, irregular cycles and possible increased risk of anovulation. [START_REF] Windham | Cigarette smoking and effects on menstrual function[END_REF] Thus, it could be that smokers misattribute cycle irregularity to the vaccine rather than to smoking if they are more attentive to their cycles after vaccination because they already experience irregular cycles. Alternatively, smoking could impact vaccine side-effects more generally through its impact on the immune system, although there is no link published on J o u r n a l P r e -p r o o f vaccine side-effects and smoking. Yet, given that smoking induces systemic chronic inflammation, smokers may be at an increased risk of menstrual cycle disturbance due to an exacerbation of inflammation following vaccination against COVID-19.

Our study shows no association between the brand of vaccine (Pfizer vs. AstraZeneca) nor the number of doses (1 vs. 2) with post-vaccination menstrual changes. This result is in line with reports made on the Yellow Card surveillance scheme reporting, and with other studies comparing menstrual changes following the Pfizer and Moderna vaccines, [START_REF] Lee | Characterizing menstrual bleeding changes occurring after SARS-CoV-2 vaccination[END_REF] or between the Pfizer, AstraZeneca and Moderna vaccines. [START_REF] Male | Effect of COVID-19 vaccination on menstrual periods in a retrospectively recruited cohort[END_REF] The absence of any association between pre-existing reproductive conditions and selfreported changes partly differs from the findings of other studies. In a previous UK study, participants with PCOS and endometriosis were "somewhat" more likely to report, respectively, a later and earlier timing of cycle after vaccination (borderline significance), but participants with a pre-existing diagnosis of fibroids and heavy menstrual bleeding were not more likely to report a change in flow as compared to others. [START_REF] Male | Effect of COVID-19 vaccination on menstrual periods in a retrospectively recruited cohort[END_REF] Conversely, in the US study, [START_REF] Lee | Characterizing menstrual bleeding changes occurring after SARS-CoV-2 vaccination[END_REF] participants diagnosed with fibroids were slightly more likely to experience heavier bleeding. Altogether, the findings indicate that there are no strong associations between pre-existing gynaecological conditions and menstrual cycle changes.

COVID-19 disease and risk of 'abnormal' cycle parameters

The results from our analyses suggest that SARS-CoV-2 infection is potentially more concerning than COVID-19 vaccine for causing menstrual cycle changes categorized J o u r n a l P r e -p r o o f as 'abnormal' in the FIGO System of nomenclature for abnormal uterine bleeding. [START_REF] Munro | The two FIGO systems for normal and abnormal uterine bleeding symptoms and classification of causes of abnormal uterine bleeding in the reproductive years: 2018 revisions[END_REF] While participants who are vaccinated do not experience more abnormal cycle parameters than unvaccinated participants during the pandemic, a history of COVID-19 disease was associated with an increased tendency of reporting frequent cycles (<24 days), periods stopping and long period duration (8+ days), and a significant increased risk of reporting heavier flow and inter-menstrual bleeding. Those outcomes may result from various causes including ovarian irregularities, uterine issues, inflammation and hormonal imbalances. For instance, frequent cycles may suggest anovulatory cycles, short luteal phase (<10 days) and low progesterone levels, which may compromise fertility in the subsequent cycle immediately following the short luteal phase. [START_REF] Crawford | Prospective evaluation of luteal phase length and natural fertility[END_REF] To date, there is no evidence that a history of asymptomatic or mild SARS-CoV-2 infection leads to negative outcomes of IVF treatments, [START_REF] Wang | Investigating the impact of asymptomatic or mild SARS-CoV-2 infection on female fertility and in vitro fertilization outcomes: A retrospective cohort study[END_REF][START_REF] Youngster | IVF under COVID-19: treatment outcomes of fresh ART cycles[END_REF][START_REF] Setti | First trimester pregnancy outcomes in a large IVF center from the Lombardy County (Italy) during the peak COVID-19 pandemic[END_REF] but results from IVF cannot be generalizable to populations without a history of Infertility or with severe COVID symptoms. This study also found that a history of COVID-19 disease increases the risk of reporting "missing" or "stopped" periods. This association must be interpreted with caution because the variable does not map onto the medical definition of amenorrhea (cessation of previously regular menses for 3 months) and merely captures participants' perception. Yet, this finding echoes a recently published case of secondary amenorrhea following SARS-CoV-2 infection in a 36-year-old healthy woman, suggesting greater attention should be focused on SARS-CoV-2-induced hypothalamic-pituitary dysfunction. [START_REF] Facondo | Case Report: Hypothalamic Amenorrhea Following COVID-19 Infection and Review of Literatures[END_REF] As compared to individuals who are vaccinated, a history of COVID-19 disease is significantly associated with an increased risk of reporting more inter-menstrual bleeding and heavier bleeding during the pandemic, which is in line with previous studies showing an association between abnormal uterine J o u r n a l P r e -p r o o f bleeding and both subclinical Chlamydia infection [START_REF] Toth | Association Between Chlamydia trachomatis and Abnormal Uterine Bleeding[END_REF] and dengue fever [START_REF] Tangnararatchakit | Excessive Menstrual Bleeding in Adolescents With Dengue Infection[END_REF] . There is currently limited data on the associations between COVID-19 disease and human reproduction beyond the effect of SARS-CoV-2 infection during pregnancy and IVF treatments [START_REF] Setti | First trimester pregnancy outcomes in a large IVF center from the Lombardy County (Italy) during the peak COVID-19 pandemic[END_REF] . The results here suggest that a history of COVID-19 disease can, in some cases, lead to abnormal cycle parameters, whereas receiving a COVID-19 vaccine does not. This is in line with a recent study showing a relationship between SARS-CoV-2 antibodies and menstrual irregularities [START_REF] Cherenack | Infection with SARS-CoV-2 is associated with menstrual irregularities among women of reproductive age[END_REF] .

Unanswered questions and future research

The association between a history of SARS-CoV-2 infection and menstrual disturbances post-vaccination in this study may be partly due to the effect of prior infection with SARS-CoV-2 on the immune response to vaccination, which has been found to be heightened [START_REF] Kelsen | Heightened COVID-19 Vaccine Response Following SARS-CoV-2 Infection[END_REF] . Biological data would be needed to verify this hypothesis.

Our findings also suggest that exogenous oestrogen may reduce post-vaccination menstrual disturbances through anti-inflammatory or anti-viral effects. This is consistent with the recent suggestion that an 'inflammatory' rather than an 'ovulatory' route might explain menstrual disturbances following COVID-19 vaccination given the high prevalence of breakthrough bleeding among users of long-acting reversible contraceptives (LARC) [START_REF] Lee | Characterizing menstrual bleeding changes occurring after SARS-CoV-2 vaccination[END_REF] . A protective effect of oestrogen [START_REF] Haitao | COVID-19 and Sex Differences[END_REF] and oestradiol [START_REF] Costeira | Estrogen and COVID-19 symptoms: Associations in women from the COVID Symptom Study[END_REF]

Limitations of the study

Our analysis uses data from a survey not specifically designed to investigate the impact of COVID-19 vaccination on menstruation. It is retrospective in nature as well as sensitive to selection, recall and report biases, and does not systematically assess the full spectrum of menstrual disturbance defined by the International Federation of Gynaecology and Obstetrics Abnormal Uterine Bleeding System 1 [START_REF] Munro | The two FIGO systems for normal and abnormal uterine bleeding symptoms and classification of causes of abnormal uterine bleeding in the reproductive years: 2018 revisions[END_REF] . For instance, we cannot speak to abnormal uterine bleeding for heavy bleeding as the question was drafted in terms of changes (heavier). We took several steps to limit selection bias during sampling (see methods) and the initial survey is broadly representative of people infected with COVID in the UK (8.9% with a positive PCR test in our study compared to a national proportion of 6.6% at the time [START_REF] Government | Coronavirus Cases in the United Kingdom[END_REF] ). However, approximately 45% of the sample had received at least one dose of the vaccine, as compared to the national proportion of 59% by the time of the last survey entry [START_REF]COVID-19) in the UK: Vaccinations in United Kingdom[END_REF] . In addition, menstrual changes may manifest later after vaccination, and our study does not have the time depth to evaluate this possibility. Among studies of other vaccines conducted on a longer timescale, no effect was found by 6-9 months [START_REF] Lamb | Experiences with prophylactic Typhoid vaccination[END_REF][START_REF] Shahani | Evaluation of endocrine parameters in clinical trials with β-hCG vaccine[END_REF] .

J o u r n a l P r e -p r o o f

The history of COVID-19 disease in our study is self-reported and there are no biological data to confirm diagnosis. Therefore, there might be a number of asymptomatic individuals in our study population who may not have reported a history of COVID-19 disease although they were infected. However, our results are conservative because this bias would have reduced, rather than increased, differences between the groups of interest. Further, we are unable to fully ascertain that it is the virus, rather than its impact on people's lives, that is causing the associations, yet the associations between vaccination and menstrual changes remain after adjusting for changes in eating behaviour and physical exercise (analyses not shown). Finally, we are unable to evaluate if such changes are decreased or increased by vaccination (most individuals in the sample were likely vaccinated after COVID-19 disease rather than the other way around), if they are temporary or last in time, and the risk factors for experiencing menstrual cycle changes after infection. Yet, our findings point to the importance of routine assessment of reproductive health and time of last menstrual period as part of the health assessment of women with an infection.

The survey is sensitive to recall bias, although this bias is limited compared to more recent surveys because sampling was conducted before widespread media attention to the topic [START_REF] Trogstad | Increased Occurrence of Menstrual Disturbances in 18-to 30-Year-Old Women after COVID-19 Vaccination[END_REF][START_REF] Lee | Characterizing menstrual bleeding changes occurring after SARS-CoV-2 vaccination[END_REF][START_REF] Male | Effect of COVID-19 vaccination on menstrual periods in a retrospectively recruited cohort[END_REF] : the issue of menstrual disturbances was not reported by the British Broadcasting Corporation until May 13, 2021 [START_REF] Robinson | Covid vaccine: Period changes could be a short-term side effect[END_REF] , as compared to a flurry of attention in US media throughout April [START_REF] Mcshane | Can the vaccine make your period worse? These women say yes[END_REF][START_REF] Saar | Women Say COVID Vaccine Side Effects Impact Their Periods, So Why Don't Doctors Care? Haaretz[END_REF][START_REF] Efrati | Some Vaccinated Israeli Women Report Irregular Menstrual Cycles[END_REF] . Further, we obtained the same results when we restricted the analysis to participants who completed the survey before the month of April 2021, suggesting our findings are less likely to be driven by individuals exposed to the idea of vaccine-related menstrual disturbances on social media. Finally, J o u r n a l P r e -p r o o f compared to previous studies investigating both vaccination and infection [START_REF] Wang | Investigating the impact of asymptomatic or mild SARS-CoV-2 infection on female fertility and in vitro fertilization outcomes: A retrospective cohort study[END_REF] , this study is better powered to compare vaccination and infection.

Inclusion and diversity

We worked to ensure gender balance, ethnic or other types of diversity in the recruitment of human subjects. We worked to ensure that the study questionnaires were prepared in an inclusive way. One or more of the authors of this paper selfidentifies as an underrepresented ethnic minority in their field of research or within their geographical location. A prolonged period is defined as >8 days. The predicted probability to report long periods is higher in the Cov group compared to the Vax group (+65%). (F) Period "missed". Participants were asked whether they perceived having missed a period or whether their periods had stopped. The probability of reporting periods "stopping" or "missed" is higher in the Cov group compared to the Vax group (+31%). J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f

J o u r n a l P r e -p r o o f

Self-diagnosed + 208 (0) 0 (0) 5,788 (100) 3,635 (100)
advertising campaign targeting all menstruators in the UK, and included images of women of diverse ethnicities, ages, and abilities, as well as images of breastfeeding and pregnant women; The title of the survey was kept general ("women's reproductive health and the COVID pandemic") so as not to oversample individuals with specific interest in menstrual cycles and COVID infection or vaccination. We fine-tuned the ad targeting (to the extent that Facebook allows) throughout the campaign to ensure even geographical and socio-economic spread. We also used a stratified sampling strategy to ensure that subgroups of the UK population in terms of age, income and ethnicity were represented in the final sample. In total, 695,543 people viewed the survey ad on their Facebook page and 26,710 with eligible criteria gave consent and completed it (there were no duplicates), leading to a 3.8% response rate. In this sample, participants were aged 18-45, 95% identified as White ethnicity and 99% identified as women.

Method details Survey design

Our online survey was designed to evaluate whether and how the COVID-19 pandemic influenced menstrual health. During the design of survey questions, input from a panel of women suffering from Long Covid, referred to us by the Long Covid Support (https://www.longcovid.org/), was incorporated. Retrospective and self-reported data on menstrual cycles, behaviour, life circumstances and health before and during the pandemic as well as COVID-19 disease and vaccination history were collected using an online survey hosted on the Qualtrics platform (www.qualtrics.com). All survey responses were anonymized using randomly generated IDs.

J o u r n a l P r e -p r o o f

The online survey was launched on March 8, 2021. The survey included a maximum of 105 questions depending on individual circumstances and took an average of 24 minutes to complete. Of the eligible participants who started the survey, 61% answered all questions after giving their consent (on average participants completed 80% of the questionnaire). In case of survey fatigue, progress could be saved for up to 14 days to allow participants to resume later. The survey ran from 08/03/21 to 01/06/21 and was closed when there had been no new entries for a week.

Outcome variables

Objective 1: Perceived vaccine side-effects on menstrual cycles

While the survey did not initially aim to evaluate the impact of vaccination on menstrual cycles specifically, a question was included to assess participants' perception of their menstrual cycles following vaccination at the end of the survey. Specifically, participants who indicated that they had been menstruating in the past 12 months, received 1 or 2 doses of the COVID-19 vaccines and were not involved in a clinical trial were asked "Have you noticed any changes to your menstrual cycles since you got vaccinated?", to which 1 of 4 possible answers could be given: "No", "Yes, my menstrual cycles are MORE disrupted", "Yes, my menstrual cycles are LESS disrupted", "Other (please state)". Although "disruption" per se was not defined, by the time participants answered this question, they had already completed many questions on menstrual cycle regularity, duration, and symptoms. At the time of the survey design, anecdotal reports of menstrual effects of the vaccine were only just beginning to circulate. Participants could select the answer "Other", which in some cases may J o u r n a l P r e -p r o o f not have been a different decision from choosing either "more disrupted" or "less disrupted". For analysis, we thus transformed these variables to represent a binary outcome ("No changes" vs. "Any other changes").

Objective 2: Menstrual parameters

We operationalized our outcome variables to approximate the FIGO classification system for normal and abnormal uterine bleeding in relation to 5 parameters: frequency, regularity, duration, volume, and inter-menstrual bleeding (FIGO System 1, [START_REF] Munro | The two FIGO systems for normal and abnormal uterine bleeding symptoms and classification of causes of abnormal uterine bleeding in the reproductive years: 2018 revisions[END_REF] ).

Frequency

In the later part of the survey, participants were asked "Over the last year, how many days long, on average, was your cycle (between the start of one bleed, and the start of the next bleed)?". Based on the number of days reported, we created a variable with 3 possible outcomes (Normal [24 to 38 days], Frequent [<24 days],

Infrequent [>38 days], based on FIGO definitions).

Participants were also asked "Over the last year, have your periods stopped?" and "Over the last year, did you miss your periods at least once?" Although "stop" and "miss" were not defined, concerns over "missing periods" were being reported on social media and thus this variable was meant to capture people's perception of their cycles from which we created a binary variable (perception of 'missing' or 'stopped' periods (0/1)).

Regularity

Participants were asked "Over the last year, how irregular was the length of your menstrual cycles on average?". We created a variable with 3 possible outcomes Volume. "Over the last year, have you noticed any changes in your periods?" There were 4 possible outcomes ("Heavier", "Lighter", "No Changes" and "Heavier and

Lighter").

Inter-menstrual bleeding Over the last year, have you noticed any changes in

spotting mid-cycle? There were 4 possible outcomes ("No changes", "More", "Sometimes", "Sometimes less and sometimes more".

Exposures

A total of 33 variables were extracted for this analysis.

Quantification and statistical analysis

We restricted all analyses to pre-menopausal individuals living in the UK who had a period in the 12 months preceding the survey and who were not pregnant or breastfeeding. Further, we only included individuals who knew their COVID-19 disease and vaccination history at the time of the survey. In the sample, most individuals selfidentify as white (95%) and as women (99%). We then grouped categories for the variables gender (women vs. other) and ethnic group (white vs. other) in univariable Our main exposure variable described participants' self-reported COVID-19 disease and vaccination history and had 4 levels (1) vaccinated but not infected; (2) vaccinated and infected (unknown order); (3) infected only and (4) neither vaccinated nor infected.

Our referent group was "vaccinated only". We used multinomial models when the outcome variables were nominal (two or more categories with no intrinsic order) and log-binomial regressions when the outcome was dichotomous. To evaluate changes between menstrual cycle characteristics, we adjusted all models for menstrual characteristics before the pandemic, and included age, BMI, hormonal contraceptive use and presence of reproductive disease at baseline as confounders as per hypothesized directed acyclic graphs (Figure S6). Estimates and confidence intervals on the log-odds scale were converted to relative risk ratios (multinomial models) and those on the log-probability scale (log-binomial models) were converted to prevalenceratios for reporting in tables and figures. To investigate if any associations between our exposure variable and menstrual cycle changes were influenced by confounders, we compared models with and without interaction effects using AIC. We reported variables significant at the false discovery rate (FDR) threshold (FDR-corrected P<0.05) 53

Missing data

The analysis of complete cases only by dropping missing cases can introduce bias and lead to a substantial reduction of statistical power [START_REF] Sterne | Multiple imputation for missing data in epidemiological and clinical research: potential and pitfalls[END_REF] , especially if it is plausible that the data are not missing at random or not completely at random. An evaluation of the missing data suggested that multiple imputation was advisable (Figure S7). The J o u r n a l P r e -p r o o f average proportion of missing values across all variables in the dataset was 3.8%, which was mostly accounted for by the variable BMI (38% of missing data, Figure S5).

To handle missing data, we used a multiple imputation approach using the R package 'missRanger' [START_REF] Mayer | missRanger: Fast Imputation of Missing Values[END_REF] , which combines random forest imputation with predictive mean matching [START_REF] Mayer | missRanger: Fast Imputation of Missing Values[END_REF] . Prior to all analyses, we imputed 5 datasets, with a maximum of 10 iterations specified for each imputation. Each imputation was also weighted by the degree of missing data for each participant, such that the contribution of data from participants with higher proportions of missingness was weighted down in the imputation. We set the maximum number of trees for the random forest to 200 but left all other random forest hyperparameters at their default. The average out-of-bag (OOB) error rate for multiple imputation across all imputed datasets was 0.08 (range: 0 to 0.77). Parameter estimates for all five datasets were pooled to provide more accurate estimates. A sensitivity analysis was also performed on the complete cases without missing data imputation (Objective 1: n=1,548; Objective 2: n=936 to n=4,862, Table S2).

Text analysis

We first built a custom text cleaning function using the 'textclean' 57 and 'tidytext' 58 R packages to analyse the text written by participants selecting the "Other" category in the outcome variable (n=574). The resulting corpus was tokenized (broken into individual units) and lemmatized (words derived from others, such as "vaccine" and "vaccination" were grouped by their stem version "vaccine"). J o u r n a l P r e -p r o o f

(

  PR=0.57, 95CI=[0.43 to 0.75], FDR P-value = 0.0002) while current smokers are 1.3 times as likely to report any changes (PR=1.31, 95CI=[1.1 to 1.58], FDR P-value = 0.006) and individuals with a positive COVID-19 disease history are 37 to 46% as likely to report menstrual changes post-vaccination [Long Covid (PR=1. 46, 95CI=[1.22 to 1.75], FDR P-value = 0.00009), acute COVID-19 (PR=1.40; 95CI=[1.20 to 1.62], FDR P-value=0.00003); self-diagnosed positive (PR=1.50, 95CI=[1.25 to 1.80], FDR Pvalue = 0.00005), tested positive (PR=1.37, 95CI=[1.16 to 1.62], FDR P-value = 0.0008, Figure 3, Table

  : (1) participants vaccinated with 1 or 2 doses but without a history of COVID-19 disease (Vax, n=3,635, 29%); (2) participants previously diagnosed with COVID-19 disease and vaccinated (Covax, n=1,354, 11%); (3) unvaccinated participants previously diagnosed with COVID-19 disease (Cov, n= 1,802, 14%); (4) Participants neither vaccinated nor previously diagnosed with COVID-19 disease at the time of the survey (None, n=5,788, 46%). The relationships between cycle parameters and the history of COVID-19 disease and vaccination are adjusted for relevant cycle parameters before the pandemic, age, BMI, contraceptive use, and reproductive disease at baseline (Table

(

  Cov: RRR = 1.3, 95CI = [1.06 to 1.6], FDR P-value = 0.050; Covax: OR = 1.32, 95CI = [1.06 to 1.64], FDR P-value = 0.052), the probability of reporting frequent cycles increasing from 26% in the vaccinated-only group to 34% in the COVID-19 disease groups. There are no significant differences between the vaccinated-only group and the COVID-19 disease-only group (Cov: RRR=1.06; 95CI = [0.91 to 1.25], FDR P-value = 0.618).

Discussion

  There has been public concern over the possibility that vaccination against COVID-19 leads to changes in menstrual cycles. Counselling women who are considering vaccination against COVID-19 thus requires identifying the risk factors for experiencing menstrual cycle changes following COVID-19 vaccination, as well as information on the relative risk of vaccines versus infection with SARS-CoV-2 for driving menstrual cycle changes. Using data collected in the UK prior to widespread media attention to menstrual disturbances following COVID-19 vaccination, this study found that (1) perceived menstrual cycle changes following vaccination are 'very common' given international pharmacovigilance standards (i.e. over 10%), (2) these perceived menstrual cycle changes are increased for participants reporting a history of COVID-19 disease, but decreased among those who use combined contraceptives, (3) vaccination alone does not lead to abnormal cycle parameters as defined by FIGO, but a history of COVID-19 disease is associated with an increased risk of reporting frequent cycles (<24 days), prolonged periods (>8 days), heavier period flow and more inter-menstrual bleeding and, (4) experiences of cycle changes after COVID-19 vaccination are diverse, including light and heavy bleeding as well as early and late periods. The results have implications for evidence-based counselling tailored to individual circumstances.

  has been suggested in relation to the severity of COVID-19, and randomized control trials on unbiased samples would be needed to establish causality between oestrogen and the reduced risk of menstrual disturbances following COVID-19 vaccination. Finally, the diversity of menstrual responses to COVID-19 vaccination might be partly explained J o u r n a l P r e -p r o o f by the timing of vaccination in relation to the menstrual cycle. An analysis of the Apple Women's Health Study found that vaccination during the follicular phase was associated with longer cycles, while a second dose of an mRNA vaccine in the luteal phase was associated with slightly shorter cycles 26 . The findings thus call for routine menstrual data collection in COVID-19 and vaccination studies as well as research into the mechanisms of menstrual disturbance following vaccination.

Figure titles and legends Figure 1 .

 1 Figure titles and legends

Figure 2 .

 2 Figure 2. Prevalence-ratios from univariable analyses of the relationship

Figure 3 .

 3 Figure 3. Predicted probability of reporting any menstrual changes following

Figure 4 .

 4 Figure 4. Flowchart of the sample selection for vaccinated and unvaccinated

Figure 5 .

 5 Figure 5. Predicted probabilities for cycle characteristics "during the pandemic"

  J o u r n a l P r e -p r o o f Vax: participants vaccinated with 1 or 2 doses but without a history of COVID-19 disease; Covax: participants diagnosed with SARS-CoV-2 infection and vaccinated; Cov: unvaccinated participants diagnosed with history of COVID-19 disease; None: participants neither vaccinated nor diagnosed with SARS-CoV-2 infection. (A) Cycle Frequency: Normal: between 24-38 days; Frequent: <24 days; Infrequent: >38 days. The probability of reporting frequent cycle vs. normal cycles increases is higher in the Cov and Covax groups than in the Vax group (+30%). (B) Cycle Regularity. Regular (less than 10 days difference between the lengths of two cycles). Cycle regularity does not vary across groups. (C) Period Flow. The probability of reporting heavier flow is higher in the Cov and Covax groups compared to the Vax group (+38%), while the probability of reporting lighter vs. normal flow is higher in the Covax compared to the Vax group (+29%). (D) Inter-menstrual bleeding. The predicted probability of reporting more inter-menstrual bleeding is higher in the Cov and Covax groups compared to the Vax group (+31%). (E) Period Duration.

Figure 6 .

 6 Figure 6. Most common words (unigrams) and pairs of adjacent words (bigrams)

Figure 7 .Figure 8 .

 78 Figure 7. Correlation matrix between key words within sentences describing

( 34 Duration

 34 Normal [>2 days; 2-5 days; 5-10 days], Somewhat irregular [10-20 days], Very irregular [>20 days]).J o u r n a l P r e -p r o o f Participants were asked "Over the last year, have you noticed any changes in the length of your menstrual cycle? Days of bleeding (Period length)" We created a binary variable with 2 possible outcomes (Normal ≤8 days; Prolonged >8+ days]).

J o u r

  n a l P r e -p r o o f Are COVID-19 vaccination and COVID-19 disease risk factors for 'abnormal' menstrual parameters? (Objective 2)

30 JHighlights•

 30 The corpus was analysed to answer the following three questions: (i) which single words (unigrams) and pairs of adjacent words (bigrams) are most frequent? (ii) which words co-occur in the same sentence? (iii) Are there clusters of symptoms? To investigate the commonality of J o u r n a l P r e -p r o o f missed J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f o u r n a l P r e -p r o o f Menstrual disturbances were reported by 1 in 5 people after COVID-19 vaccination • Perceived vaccine-related menstrual changes decreased with combined contraceptives • Vaccinated individuals were not at increased risk of abnormal uterine bleeding • COVID-19 disease associated with heavier menstrual flow volume J o u r n a l P r e -p r o o f KEY RESOURCES

Table 1 .

 1 Characteristics of the sample of vaccinated individuals

	Characteristic	N = 4,989
	Age, Median (IQR)	35 (28 -43)
	Body Mass Index, n (%)
	Healthy weight	1,059 (34)
	Obese	1,163 (37)
	Overweight	836 (27)
	Underweight	49 (1.6)
	Unknown	1,882
	Hormonal contraceptive use at the
	time of the survey, n (%)
	Combined oestrogen-progestin	441 (11)
	Copper IUD	225 (5.4)
	None	2,421 (58)
	Other	84 (2.0)
	Progestogen-only	854 (21)
	Sterilization	130 (3.1)
	Unknown	834
	COVID-19 disease (type), n (%)
	COVID -	3,377 (75)
	Long COVID	462 (10)
	Acute COVID	687 (15)
	Unknown	463
	COVID-19 disease (diagnosis), n (%)
	Negative	3,377 (76)
	Self diagnosed +	395 (8.9)
	Tested +	671 (15)
	Unknown	546
	Number of vaccination doses, n (%)
	Yes, one dose	4,096 (82)
	Yes, two doses	893 (18)
	Vaccine type, n (%)
	Oxford-AstraZeneca	2,600 (53)
	Pfizer-BioNTech	2,335 (47)
	Unknown	54
	Timing of 1st dose, n (%)
	Before 2021	331 (6.7)
	January 2021	1,497 (30)
	February 2021	1,469 (30)
	March 2021	1,659 (33)
	Unknown	33

Table 2 .

 2 Characteristics of the sample of vaccinated and unvaccinated individuals by COVID-19 status

	Characteristic\Group	Covax 1	Cov 2	None 3	Vax 4	p-value 5
		N = 1,354	N = 1,802	N = 5,788	N = 3,635
	Age, Median (IQR)	35.00 (28.00	30.00	30.00 (24.00	35.00	<0.001
		-43.00)	(24.00 -	-37.00)	(28.00 -
			38.00)		43.00)
	Body Mass Index, n (%)					<0.001
	Healthy weight	267 (31)	458 (42)	1,689 (48)	760 (34)
	Obese	354 (42)	288 (26)	728 (21)	832 (37)
	Overweight	225 (26)	316 (29)	942 (27)	616 (27)
	Underweight	6 (0.7)	36 (3.3)	124 (3.6)	38 (1.7)
	Unknown	502	704	2,305	1,389
	Hormonal contraceptives,					<0.001
	n (%)
	Combined	120 (10)	217 (15)	768 (17)	305 (10)
	Copper IUD	58 (5.1)	87 (6.0)	257 (5.6)	169 (5.6)
	None	661 (58)	802 (56)	2,567 (56)	1,795 (59)
	Other	23 (2.0)	20 (1.4)	91 (2.0)	64 (2.1)
	Progestogen-only	257 (22)	292 (20)	861 (19)	599 (20)
	Sterilization	28 (2.4)	26 (1.8)	71 (1.5)	99 (3.3)
	Unknown	207	358	1,173	604
	COVID type, n (%)					<0.001
	Acute COVID	848 (64)	1,169 (67)	0 (0)	0 (0)
	Long COVID	475

  Unvaccinated participants with no history of COVID-19 disease;[START_REF] Lomte | Impact of SARS-CoV-2 infection on menstrual cycle[END_REF] Unvaccinated participants with a history of COVID-19 disease;[START_REF] Morgan | Pandemic Periods: WhyWomen's Menstrual Cycles Have Gone Haywire[END_REF] Kruskal-Wallis rank sum test; Pearson's Chi-squared test.

					)
	Number of doses, n (%)				<0.001
	Unvaccinated	0 (0)	1,802 (100)	5,788 (100)	0 (0)
	1 dose	1,110 (82)	0 (0)	0 (0)	3,023 (83)
	2 doses	244 (18)	0 (0)	0 (0)	612 (17)
	Vaccine type, n (%)				0.66
	Oxford-AstraZeneca	725 (54)	0 (NA)	0 (NA)	1,969 (55)
	Pfizer-BioNTech	616 (46)	0 (NA)	0 (NA)	1,626 (45)
	Unknown	13	1,802	5,788	40
	Timing 1st dose, n (%)				0.31
	Before 2021	88 (6.5)	0 (NA)	0 (NA)	227 (6.3)
	February 2021	385 (29)	0 (NA)	0 (NA)	1,034 (29)
	January 2021	412 (31)	0 (NA)	0 (NA)	1,016 (28)
	March 2021	465 (34)	0 (NA)	0 (NA)	1,330 (37)
	Unknown	4	1,802	5,788	28
	1 Participants both vaccinated and with a history of COVID-19 disease; 2 Unvaccinated participants with a history of COVID-19
	disease; 3

  acute COVID (symptoms lasting less than 28 days) and Long Covid (symptoms lasting more than 28 days; we only included people who had symptoms more than a month before taking up the survey) as well as (ii) based on a combination of testing and selfdiagnosis, leading to three categories: No COVID (no tests or negative tests), COVID tested + (positive test) and "Self-diagnosed positive" (referring to individuals who had a suspected or clinically diagnosed COVID infection but had not obtained positive PCR, antigen or antibody tests). We included this last category due to the unavailability of widespread testing in the UK in the first wave of the pandemic in 2020 and ongoing questions about the accuracy and optimal timing of antigen and antibody tests. Third,

	standard proxies for health (BMI, smoking status, physical activity, regular use of
	vitamins/supplements, regular use of medicine) and reproductive variables indicative
	of menstrual health before the pandemic (age at menarche, cycle length, period length,
	cycle irregularity, heavy bleeding and contraceptive use), the dataset included
	vaccine-related, COVID and pandemic-related variables. First, data on the type of
	vaccine received, of which only two had been approved for use in the UK at the time
	(Pfizer BioNTech/Oxford-AstraZeneca/Not sure), and the timing of the first vaccination
	(month/year) were included. Second, COVID-19 disease was operationalized in two
	ways: (i) based on whether people thought they had had COVID, as widespread testing
	had not been available in the UK in the early months of the pandemic which fell within

In addition to socio-demographic variables (age, income, education, gender, ethnic group, marital status, parity), the survey period, leading to three categories: No COVID (no tests or negative tests), J o u r n a l P r e -p r o o f hormonal contraceptive use was categorized as progestogen-only (hormonal coil or IUS, implant, injectable, progestogen-only pill), combined oestrogen and progestin (the pill, the patch, vaginal ring), copper IUD, sterilization, none (fertility awareness, condom, female condom, diaphragm) and other. Fourth, a variable indicative of changes in life satisfaction compared to before the pandemic was included to adjust for changes experienced because of the pandemic and/or the infection rather than vaccination.

TABLE

  ). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/.

	REAGENT or RESOURCE	SOURCE	IDENTIFIER
	Deposited Data
	Data and scripts Open Science	DOI 10.17605/OSF.IO/PQXY2
		Framework
	Data collection	Qualtrics XM	www.qualtrics.com
	platform
	Software and Algorithms
	R version 4.2.2 The R Project for	Core Team (2020
		Statistical
		Computing

Acknowledgments

We thank participants for their time completing the survey. JAM receives funding from The Wellcome Trust (209589/Z/17/Z, IS3-R3.11 21/22) and The Royal Society of Edinburgh (1077) and acknowledges the support of MRC Centre Grants G1002033 and MR/N022556/1. AA received funding from the British Academy (MD19\190016). GK received funding from the ESRC ES/P000649/1.

Author contributions

AA, GK and ZO supervised the entire study, designed the survey, and wrote the original draft of the manuscript; GK conducted the text analysis; AA conducted the quantitative analysis and revised the manuscript; GS, MAA and JAM provided intellectual contributions to the survey design and analysis and revised the manuscript.

LA, NR and DK provided patient feedback on the design of the survey and revised the manuscript.

Declaration of interests

The authors declare no competing interests.

J o u r n a l P r e -p r o o f

STAR★Methods Resource availability

Lead contact: Further information and requests for data and scripts should be directed to and will be fulfilled by the lead contact, Alexandra Alvergne (alexandra.alvergne@umontpellier.fr). Participants could only complete the survey if they were over 18, had ever menstruated, currently lived in the UK, and gave informed consent to the use of their data. The survey was written in English and disseminated through a Facebook J o u r n a l P r e -p r o o f analyses. We then applied several additional exclusions depending on the analysis.

Materials availability:

We reported prevalence-ratios and relative risk ratios in the text, and plotted predicted probabilities from adjusted models to represent absolute effects adjusted for confounders.

What are the risk factors for perceiving menstrual cycle changes following COVID-19 vaccination ? (Objective 1)

We first conducted a series of exploratory univariable analyses, investigating each of the 33 variables in relation to menstrual characteristics during the pandemic. We then retained all variables significant at the false discovery rate (FDR) threshold (FDRcorrected P<0.05) [START_REF] Benjamini | Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing[END_REF] for consideration in multivariable analyses. We then conducted multivariable analyses for each potential risk factor adjusting for potential confounders, which were defined as variables significant in the univariable analyses and with a potential confounding (but not mediating) effect according to hypothesized directed acyclic graphs (Figure S1, Figure S2, Figure S3, Figure S4, Figure S5, Figure S6).

Because the original outcome variable was nominal (two or more categories with no intrinsic order) but violated the IIA assumption (Independence or Irrelevant Alternatives) as options were not independent, we dichotomized the variable into two mutually exclusive categories ("No changes", "Any other changes") and performed logbinomial regressions, which are appropriate when the outcome is not rare (prevalence >10%) [START_REF] Barros | Alternatives for logistic regression in cross-sectional studies: an empirical comparison of models that directly estimate the prevalence ratio[END_REF] . Exponentiating the coefficients result in prevalence ratios (PR) displayed in tables and figures.

J o u r n a l P r e -p r o o f words, we explored the frequency of unigrams and bigrams within all responses. We performed a correlation analysis on the most important words for menstrual cycle descriptions to measure the association between words using the correlation index (phi coefficient (φ) displayed in Figure 7). To explore patterns of symptoms we examined which words commonly occur together (though not necessarily adjacent) to visualize groups of words that cluster together. Clusters were visualized by arranging correlated words into a combination of connected nodes (network graph) using the 'igraph' package [START_REF] Csardi | The Igraph Software Package for Complex Network Research[END_REF] .

J o u r n a l P r e -p r o o f

Supplemental information titles and legends

Table S1: Models output related to Figures 2 and3.

Table S2: Complete cases analyses related to Figures 2 and3 Table S3: Model outputs related to Figure 5 Table S5: Complete cases analyses related to Figure 5 J o u r n a l P r e -p r o o f


Cyril Courtial
email: cyril.courtial@laposte.net

Kaïna Privet
email: kaina.privet@univ-rennes1.fr

Xavier Aubriot
email: xavier.aubriot@universite-paris-saclay.fr

Lionel Picard
email: lionel.picard@outlook.fr

Julien Petillon
email: julien.petillon@univ-rennes1.fr

Description of a new species of Hypaeus (Araneae: Salticidae: Salticinae: Amycini) based on integrative taxonomy

Keywords: Amycini) based on integrative taxonomy jumping spiders, barcoding, cytochrome c oxydase 1 subunit 1, taxonomy, French Guiana

Introduction

Neotropical spider faunas are still largely understudied (e.g. Dupérré, 2022), owing to their high diversity [START_REF] Cardoso | Global Patterns of Guild Composition and Functional Diversity of Spiders[END_REF][START_REF] Privet | Spider assemblage structure in a neotropical rainforest-inselberg complex: ecological and methodological insights from a small-scale intensive survey[END_REF] and the high human and material costs to study them. Additionally, large proportions of rare species and juveniles make up tropical spider assemblages [START_REF] Coddington | Undersampling bias: the null hypothesis for singleton species in tropical arthropod surveys[END_REF], being difficult or impossible to identify and describe. Spiders are a model group for ecological studies [START_REF] Birkhofer | The global relationship between climate, net primary production and the diet of spiders[END_REF][START_REF] Cardoso | Global Patterns of Guild Composition and Functional Diversity of Spiders[END_REF][START_REF] Malumbres-Olarte | The same but different: equally megadiverse but taxonomically variant spider communities along an elevational gradient[END_REF][START_REF] Moya-Laraño | Linking traits, selective pressures and ecological functions[END_REF]) and the only group to be exclusively predatory among the dominant terrestrial arthropods [START_REF] Birkhofer | The global relationship between climate, net primary production and the diet of spiders[END_REF][START_REF] Pekár | Trophic specialisation in a predatory group: the case of prey-specialised spiders (Araneae): Prey-specialised spiders (Araneae)[END_REF]. Improving knowledge on their taxonomy is thus crucial to understand neotropical diversity and ecology.

Within the forests of the Amazon, the most abundant and speciose spiders are the jumpingspiders (Salticidae Blackwall,1841;[START_REF] Bodner | The biogeography and age of salticid spider radiations (Araneae: Salticidae)[END_REF] and the richest subfamily is Amycinae [START_REF] Araújo | Description of four new species of Hypaeus Simon and redescription of H. taczanowskii Mello-Leitão (Araneae: Salticidae: Amycinae)[END_REF][START_REF] Maddison | A phylogenetic classification of jumping spiders (Araneae: Salticidae)[END_REF]. Among them, Hypaeus Simon 1900 is one of the richest genera with 28 described species distributed from south to central America (World Spider Catalog 2021).

Species of Hypaeus are distinguished by: high carapaces, plumose setae over front eyes, pluridentate chelicerae with 4-5 teeth on promargin and 3-6 on retromargin, mastidia on male ACCEPTED MANUSCRIPT / CLEAN COPY chelicerae and third legs longer than fourth [START_REF] Galiano | Revision de los géneros Acragas, Amycus, Encolpius, Hypaeus, Mago y Noegus (Salticidae, Araneae)[END_REF]Ruiz & Madisson 2015). However, Hypaeus is still poorly described with inadequate sampling across the genus' range. Thus, the boundaries of Hypaeus are not completely understood [START_REF] Araújo | Description of four new species of Hypaeus Simon and redescription of H. taczanowskii Mello-Leitão (Araneae: Salticidae: Amycinae)[END_REF]. [START_REF] Galiano | Las especies americanas de arañas de la familia Salticidae descriptas por Eugène Simon: Redescripciones basadas en los ejemplares típicos[END_REF][START_REF] Galiano | Revision de los géneros Acragas, Amycus, Encolpius, Hypaeus, Mago y Noegus (Salticidae, Araneae)[END_REF]) redescribed and revised 18 Hypaeus species described by [START_REF] Crane | Spiders of the families Lyssomanidae and Salticidae from British Guiana and Venezuela[END_REF], [START_REF] Mello-Leitão | Contribuição ao conhecimento da fauna araneológica das Guianas[END_REF], [START_REF] Peckham | On some new genera and species of Attidae from the eastern part of Guatamala[END_REF], [START_REF] Simon | Etudes arachnologiques. 30e Mémoire. XLVII. Descriptions d'espèces nouvelles de la famille des Attidae[END_REF] and [START_REF] Taczanowski | Les aranéides de la Guyane française[END_REF][START_REF] Taczanowski | Les Aranéides du Pérou. Famille des Attides[END_REF]. She also transferred two species to this genus, Amycus mystacalis [START_REF] Taczanowski | Les Aranéides du Pérou. Famille des Attides[END_REF] and Triptolemus benignus [START_REF] Peckham | On some new genera and species of Attidae from the eastern part of Guatamala[END_REF]; and synonymized one, Mago budoninus [START_REF] Caporiacco | Araignées de la Guyane Française du Muséum d'Histoire Naturelle de Paris[END_REF]. Two others were described by Crane, H. duodentatus [START_REF] Crane | Spiders of the families Lyssomanidae and Salticidae from British Guiana and Venezuela[END_REF]by Caporiacco, H. barromachadoi Caporiacco, 1947. Recently, the male and the female of the type species of the genus Hypaeus, H. taczanowskyii (Mello-Leitao, 1948), were redescribed by [START_REF] Araújo | Description of four new species of Hypaeus Simon and redescription of H. taczanowskii Mello-Leitão (Araneae: Salticidae: Amycinae)[END_REF]. The same authors added four new species to the genus, H. tridactylus, H. famoratus, H. poseidon and H. terramediae [START_REF] Araújo | Description of four new species of Hypaeus Simon and redescription of H. taczanowskii Mello-Leitão (Araneae: Salticidae: Amycinae)[END_REF] transferred Hasarius pauciaculeis (Caporiacco, 1947) to Hypaeus. The last issue from Martinez & Galvis (2017) added three new species, H. arhuaco, H. proszynskii and H. varzea Martinez & Galvis, 2017. Of the resulting 28 Hypaeus species, 17 have been described only from males [START_REF] Araújo | Description of four new species of Hypaeus Simon and redescription of H. taczanowskii Mello-Leitão (Araneae: Salticidae: Amycinae)[END_REF]Martinez & Galvis 2017;World Spider Catalog 2021).

Combining multiple lines of evidence such as morphology and DNA barcoding in species delimitation, could help alleviate the limitations on tropical spider diversity knowledge linked to juveniles and rare species. Phylogenetic analyses are also needed to resolve the relationships within Hypaeus (Ruiz et al. 2019). However, only sequences for two Hypaeus species, H. mystacalis [START_REF] Taczanowski | Les Aranéides du Pérou. Famille des Attides[END_REF] and H. miles [START_REF] Simon | Etudes arachnologiques. 30e Mémoire. XLVII. Descriptions d'espèces nouvelles de la famille des Attidae[END_REF], are available to date.

One hundred and two species of Salticidae are currently listed for French Guiana [START_REF] Courtial | Validation of Eustiromastix guianae (Caporiacco, 1954) (Araneae, Salticidae) with a first description of the female, and additions to ACCEPTED MANUSCRIPT / CLEAN COPY the salticid fauna of French Guiana[END_REF][START_REF] Vedel | Biodiversity baseline of the French Guiana spider fauna[END_REF]) and among them three Hypaeus species: H. flavipes [START_REF] Simon | Etudes arachnologiques. 30e Mémoire. XLVII. Descriptions d'espèces nouvelles de la famille des Attidae[END_REF], H. porcatus [START_REF] Taczanowski | Les aranéides de la Guyane française[END_REF]H. taczanowskii (Mello-Leitao, 1948). During our last ACCEPTED MANUSCRIPT / CLEAN COPY expeditions to French Guiana (see [START_REF] Privet | Spider assemblage structure in a neotropical rainforest-inselberg complex: ecological and methodological insights from a small-scale intensive survey[END_REF]Privet & Petillon 2018), in the National Nature Reserves of La Trinité and Les Nouragues, numerous Salticidae were collected, among them several specimens of an undescribed Hypaeus species. In this study, we used a combination of morphology and DNA barcoding targeting COI to match and describe the male and female from the new species, Hypaeus olympeae sp. nov., produce sequences for four other Hypaeus species never sequenced so far, and add nine species to the list of salticids from French Guiana.

Material and Methods

Taxon sampling

Taxa sequenced in this study are 12 individuals of salticids belonging to the genus Hypaeus.

They were collected in La Trinité (4°35′20′′N; 53°18′1′′W) and Les Nouragues (4°04′18′′N; 52°43′57′′W) Nature Reserves (French Guiana) during two surveys conducted in 2010 and 2013 (see [START_REF] Privet | Spider assemblage structure in a neotropical rainforest-inselberg complex: ecological and methodological insights from a small-scale intensive survey[END_REF][START_REF] Privet | Spider assemblage structure in a neotropical rainforest-inselberg complex: ecological and methodological insights from a small-scale intensive survey[END_REF]. In these reserves, seasonally flooded (Aya and Pararé) and summit inselberg (La Roche Bénitier and Nouragues) forests were sampled.

Inselbergs are rocky outcrops rising abruptly from the surrounding landscape where little research has been devoted to invertebrates, especially spiders [START_REF] Privet | Spider assemblage structure in a neotropical rainforest-inselberg complex: ecological and methodological insights from a small-scale intensive survey[END_REF]). These specimens were separated based on morphology and sequenced. Six specimens were identified as H. taczanowskii and four to H. porcatus. Two specimens, impossible to assign to any of the currently recognized Hypaeus species, correspond to the new species described here, Hypaeus olympeae sp. nov. Total DNA of the 12 Hypaeus specimens was then extracted using nondestructive method (see below).

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Specimens

The type material examined is deposited at the Museum national d'Histoire naturelle (MNHN, Paris). Preserved specimens were studied using an OlympusSZX9 stereomicroscope with a Moticam5 (5.0MP) camera. Live specimens (holotype male and partype female) were photographed with a Canon EOS 450 D digital reflex camera with a 60 mm macro lens. The epigyne was macerated in 10% KOH. The specimens were preserved in 70% ethanol.

Measurements were taken according to [START_REF] Edwards | Revision of the jumping spiders of the genus Phidippus (Araneae: Salticidae)[END_REF] in millimeters (mm), for direct comparison with Hypaeus spp. described by [START_REF] Araújo | Description of four new species of Hypaeus Simon and redescription of H. taczanowskii Mello-Leitão (Araneae: Salticidae: Amycinae)[END_REF]. The drawings of male individuals of [START_REF] Galiano | Las especies americanas de arañas de la familia Salticidae descriptas por Eugène Simon: Redescripciones basadas en los ejemplares típicos[END_REF][START_REF] Galiano | Revision de los géneros Acragas, Amycus, Encolpius, Hypaeus, Mago y Noegus (Salticidae, Araneae)[END_REF], [START_REF] Crane | Spiders of the families Lyssomanidae and Salticidae from British Guiana and Venezuela[END_REF], [START_REF] Ruiz | Redescription and resolution of some Neotropical species of jumping spiders described by Caporiacco and description of a new species (Araneae: Salticidae)[END_REF]), Araujo & Ruiz (2015) and Martinez & Galvis (2017), as well as the reviewing of type specimens stored at the MNHN (Paris, France), allowed us to identify the new species Hypaeus olympeae sp. nov. collected in French Guiana.

DNA extraction, amplification, sequencing and data assembly

ACCEPTED MANUSCRIPT / CLEAN COPY DNA was extracted using DNeasy 96 Blood and Tissue Kit (Qiagen) following manufacturer's recommendations. The entire specimens were directly placed in lysis buffer with proteinase K overnight to extract DNA without compromising morphological requirements for further examination [START_REF] Paquin | Testing compatibility between molecular and morphological techniques for arthropod systematics: a minimally destructive DNA extraction method that preserves morphological integrity, and the effect of lactic acid on DNA quality[END_REF]. The standard animal DNA barcode fragment of the mitochondrial cytochrome-c-oxidase subunit 1 (COI) was targeted by PCR using the primers C1-J-1718 "SPID" [START_REF] Simon | Evolution, Weighting, and Phylogenetic Utility of Mitochondrial Gene Sequences and a Compilation of Conserved Polymerase Chain Reaction Primers[END_REF]) and C1-N-2776 [START_REF] Hedin | A Combined Molecular Approach to Phylogeny of the Jumping Spider Subfamily Dendryphantinae (Araneae: Salticidae)[END_REF]. PCR reactions were performed in 25 µL and contained 0.5X PCR Buffer, 0.2 mM dNTPs, 1 mM MgCl2, 0.15 µM of each primer, 0.02 U/µL of Taq polymerase (GoTaq, Promega) and 2µL of DNA. PCR amplification started with an initial 94°C denaturation step for 2 min, followed by 35 cycles of (i) denaturation at 94°C for 45 s, (ii) annealing at 50°C for 45 s, and (iii) extension at 72°C for 60 s; a final 72°C extension step lasted 10 min. Sequencing of PCR products was performed by Genoscreen (Lille, France) using the same primers than for amplification.

Sequence fragments were imported, assembled, and edited in Geneious 6. 1.8 (Biomatters Ltd.,Auckland,New Zealand). All sequences, with voucher information, are archived in GenBank (Table 1).

Phylogenetic analysis

The taxonomic sampling used for the phylogenetic analyses included the 12 sequences newly generated for the study as well as one GenBank accession of Sarinda cutleri (Richman, 1965) (JX145669), a closely related species (see [START_REF] Maddison | The deep phylogeny of jumping spiders (Araneae, Salticidae)[END_REF]) that served as the outgroup.

All sequences were aligned in Geneious using MAFFT v.7.017 [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: improvements in performance and usability[END_REF].

The resulting COI matrix was subjected both to Bayesian inference (BI) and maximum likelihood (ML) analyses. We first searched for the best-fitting nucleotide substitution model using the Akaike information criterion (AIC) estimated by MrModeltest v.2.3 [START_REF] Nylander | MrModeltest v2. Computer program and documentation distributed by the author[END_REF]);

ACCEPTED MANUSCRIPT / CLEAN COPY

the GTR + G model was selected and assigned to all following phylogenetic analyses. The software MrBayes 3.2.6 [START_REF] Huelsenbeck | MRBAYES: Bayesian inference of phylogenetic trees[END_REF] and RaxML-HPC v.8.1.24 [START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF]) implemented on the CIPRES Science Gateway [START_REF] Miller | Creating the CIPRES Science Gateway for inference of large phylogenetic trees[END_REF]) platform were used to perform BI and ML analyses, respectively. MrBayes analyses constituted two independent parallel runs of four Markov chains each, implemented for one million generations and sampled every 100 generations. Adequate mixing of the Markov chains and convergence of the two runs were confirmed with Tracer v1.6 (Rambaut et al. 2014). After removing a 10% burnin,the remaining trees were used to generate a 50% Bayesian majority-rule consensus tree.

For the RaxML analyses, node support was assessed using a rapid bootstrapping algorithm with 1000 bootstrap iterations.

Results

Taxonomy

Etymology

The species is named "olympeae" to pay tribute to Olympe Delavalle, daughter of Marguerite Delavalle, the Curator of the NNR Nouragues during our survey. [START_REF] Nyffeler | An estimated 400-800 million tons of prey are annually killed by the global spider community[END_REF][15][16]. . Length of femur I 2.60, II 2.30,III 2.90,IV 2.90. Patella + tibia I 3.70,II 2.80,III 3.00,IV 3.00. Metatarsus + tarsus I 2.30,II 1.80,III 2.50,IV 2.80. Variation Female (n=6) Total length: 6.20-9.00. Carapace length: 2.70-3.90. Length of leg I 7.30-8.60.

Material examined

Holotype

Distribution

Only known from French Guiana.

Comments

The holotype male and female were caught together during mating behaviour in a building at the Nouragues field station.

DNA sequences and phylogenetic analysis

The COI alignment was 442 base pairs (bp) long and included 92 variable sites of which 72 were informative in parsimony. The BI and ML topologies are congruent; there was no topological conflict detected between the 50% Bayesian majority-rule consensus tree (Fig. 19) and the ML bootstrap 50% majority-rule consensus tree (not shown). The topology we obtained showed (1) that the two individuals of H. olympeae sp. nov. included in the study form a strongly supported monophyletic grouping (PP = 1; BS = 100), highly divergent from the two other clades in the tree, (2) that the four accessions sampled for H. porcatus are part of a strongly supported clade (PP = 1; BS = 99) resolved as sister to H. olympeae sp. nov. but with poor ACCEPTED MANUSCRIPT / CLEAN COPY support (PP = 0.81; BS = 67) and (3) that the 6 accessions identified as H. taczanowskii (3 male and 3 female individuals) were all included in a monophyletic grouping strongly supported in BI (PP = 0.99; BS = 74). The analysis of COI sequences then confirms the current morphological circumscriptions for the three Hypaeus species sampled here. Also, the branch lengths observed between the three clades recognized here are relatively high when compared with the magnitude of the divergence between the ingroup (viz. genus Hypaeus) and the outgroup (viz. Sarinda cutleri); this further supports the recognition of H. olympeae sp. nov. as a distinct species.

New records of Salticidae from French Guiana

According to [START_REF] Vedel | Biodiversity baseline of the French Guiana spider fauna[END_REF], [START_REF] Courtial | Validation of Eustiromastix guianae (Caporiacco, 1954) (Araneae, Salticidae) with a first description of the female, and additions to ACCEPTED MANUSCRIPT / CLEAN COPY the salticid fauna of French Guiana[END_REF] and [START_REF] Logunov | Taxonomic notes on the genus Lyssomanes Hentz 1845 (Araneae: Salticidae) from French Guiana[END_REF] we add nine new species for French Guiana. Corcovetella aemulatrix Galiano, 1975 Kourou, degrad saramak (5°01'N, 52°41'W), 1♀, collection date unknown, Vedel Vincent, by beating vegetation in garden near primary forest. Caporiacco, 1947 RNN Trinité, camp Aya (04°36'N, 53°24'W), 1♂, X.2010, Canard Alain, Courtial Cyril, Leroy Boris & Ysnel Frédéric, by beating the lower branches of trees. [START_REF] Simon | Etudes arachnologiques. 30e Mémoire. XLVII. Descriptions d'espèces nouvelles de la famille des Attidae[END_REF] Cyllodania fasciata (Caporicaco, 1954) syn. nov: 150, f.48, French Guiana, Saint-Jean du Maroni, 1914, Benoist leg, 1♀ examined. After examination of species described by [START_REF] Caporiacco | Araignées de la Guyane Française du Muséum d'Histoire Naturelle de Paris[END_REF] deposited in the MNHN, the type species of Cyllodania fasciata (Caporicaco, 1954), considered as a subadult by Caporiaco (1954) and [START_REF] Galiano | Nota sobre los géneros Cyllodania y Arachnomura (Araneae, Salticidae)[END_REF] is in fact an adult female of Gypogyna forceps [START_REF] Simon | Etudes arachnologiques. 30e Mémoire. XLVII. Descriptions d'espèces nouvelles de la famille des Attidae[END_REF]. Cyllodania fasciata (Caporicaco, 1954) is here presented as a new synonym of Gypogyna forceps [START_REF] Simon | Etudes arachnologiques. 30e Mémoire. XLVII. Descriptions d'espèces nouvelles de la famille des Attidae[END_REF] ACCEPTED MANUSCRIPT / CLEAN COPY Kourou, degrad saramak (5°01'N, 52°41'W), 1♀, collection date unknown, Vedel Vincent, by beating vegetation in garden near primary forest.

Eustiromastix moraballi

Pachomius nigrus

Gypogyna forceps

Marma nigritarsis [START_REF] Simon | Etudes arachnologiques. 30e Mémoire. XLVII. Descriptions d'espèces nouvelles de la famille des Attidae[END_REF] Saül (3°37'N, 53°12'W), 1♂, 08.X.2013, 1♂, 18.X.2013, Bellanger Yannick (Asper Society).

Figures 12Hypaeus olympeae sp. nov. 1 male, dorsal view, scale 0,5mm; 2. Female, dorsal view, scale 1mm.

Figures 34Hypaeus olypeae sp. nov. male. 3. Chelicerae detail (ma: mastidion), scale 0.5mm; 4. Left male palp with bump on femur (arrow), scale 1mm. Figure 19 50% majority-rule tree from the Bayesian analysis of the COI matrix. Numbers above each branch are bootstrap values > 50% followed by posterior probabilities from the Bayesian ACCEPTED MANUSCRIPT / CLEAN COPY analysis. Branch lengths are proportional to the number of substitutions per nucleotide position Figure 19 50% majority-rule tree from the Bayesian analysis of the COI matrix. Numbers above each branch are bootstrap values > 50% followed by posterior probabilities from the Bayesian analysis. Branch lengths are proportional to the number of substitutions per nucleotide position (scale bar = 0.01 substitutions).

See Table 1 for complete voucher information.

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  abbreviations are used:

  1♂ from RNN des Nouragues (4°02'N, 52°41'W), Camp Pararé, French Guiana (AR 16169), 11.XII.2013, Cyril Courtial coll. Paratypes 1♀ from RNN des Nouragues (4°02'N, 52°41'W), Camp Pararé, French Guiana (AR 16170), 11.XII.2013, Cyril Courtial coll.; 1♂, 2♀ from RNN de la Trinité (04°36'N, 53°24'W), Camp Aya, French Guiana, 08.XII.2010, Alain Canard coll. ACCEPTED MANUSCRIPT / CLEAN COPY Additional material examined French Guiana. RNN de la Trinité, Camp Aya (04°36'N, 53°24'W): 1♂, 01.XI.2008, Frédéric Ysnel coll.; RNN des Nouragues, Camp Inselberg (4°05'N, 52°41'W): 1♀, 09.XII.2013, Cyril Courtial coll.; RNN des Nouragues, Camp Pararé (4°02'N, 52°41'W): 1♀, 26.VI.2010, Vincent Vedel coll.; Montagne des Chevaux (4 43'N, 52 24'W): 1♀, 09.XI.2010, Vincent Vedel coll.; Roche Bénitier (04°36'N, 53°24'W) : 1♀, 06.IV.2010, Vincent Vedel coll. Description Male (Holotype, AR 16169) TL:6.00. CL:3.10. CW: 2.30. CH:1.80. OQL: 2.00. AERW:2.20. PERW:2.00. AMEW:0.70. CLH: 0.45. Carapace dark brown with light spot behind the fovea bearing white setae (Fig 1). Chelicera dark brown (Fig 3) with short mastidions pointed forward; 3 posterior cheliceral teeth, 2 anterior cheliceral teeth. Palp: femur curved with dorsal bump (Fig 4); tibia with quiet short rounded RTA (Figs 5, 7, 11-12); embolus emerging from proximal tegulum with median portion slightly narrowed (Figs 6, 13). Abdomen pale with median longitudinal light stripe (Figs 1, 17) and ventrally with median longitudinal dark brown stripe. Spinnerets light brown. Legs 1342 (10.00/6.90/7.80/7.50). Length of femur I2.80, II 2.20, III 2.60, IV 2.40. Patella + tibia I 4.30, II 2.70, III 2.70, IV 2.40. Metatarsus + tarsus I 2.90, II 2.00, III 2.50, IV 2.70. Variation Male (n=3) Total length: 6.00-7.10. Carapace length: 3.05-3.20. Length of leg I 9.05-10.00. Female (AR 16170) TL:9.00. CL:3.90. CW: 2.70. CH:2.00. OQL: 1.90. AERW:2.40. PERW:2.10. AMEW:0.80. CLH: 0.30. Carapace light brown with darker cephalic area, base of the cephalothorax bearing ACCEPTED MANUSCRIPT / CLEAN COPY dark spots (Fig 2). Chelicera dark brown with no mastidion; 3 posterior cheliceral teeth, 4 anterior cheliceral teeth. Epigyne with a pair of oblique COs (Figs 8-9, 14), long digitiform gland ducts and long and thin COs (Figs

  Mello-Leitao, 1940 RNN Nouragues, camp Pararé (4°02'N, 52°41'W), 1♂, 06.XII.2013, Canard Alain, Courtial Cyril, Leroy Boris, Pétillon Julien & Vedel Vincent, at sight in low vegetation. Lyssomanes tenuis Peckham & Wheeler, 1889 RNN Nouragues, camp Inselberg (4°05'N, 52°41'W), 1♂, 08.XII.2013, Courtial Cyril, by beating by beating the lower branches of trees. Scopocira abaporu Costa & Ruiz, 2014 Nouragues, Camp Pararé (4°02'N, 52°41'W), 1♂, 13.XII.2013, Courtial Cyril, by beating the lower branches of trees. Scopocira histrio Simon, 1900 ACCEPTED MANUSCRIPT / CLEAN COPY Nouragues, camp Inselberg (4°05'N, 52°41'W), 1♀, 10.XII.2013, Courtial Cyril, by beating the lower branches of trees. Nouragues, camp Pararé (4°02'N, 52°41'W), 1♂, 06.XII.2013, Canard Alain, Courtial Cyril, Leroy Boris, Pétillon Julien & Vedel Vincent, by beating the lower branches of trees. Colonus germaini Simon, 1900 Nouragues, camp Pararé (4°02'N, 52°41'W), 1♀, 12.XII.2013, Courtial Cyril, by beating the lower branches of trees.

Figures 1 - 2

 2 Figures 1 -2Hypaeus olympeae sp. nov. 1 male, dorsal view, scale 0,5mm; 2. Female, dorsale view, scale 1mm.

  Hypaeus olypeae sp. nov. male. 3. Chelicerae detail (ma: mastidion), scale 0.5mm; 4. Left male palp with bump on femur (arrow), scale 1mm.

Figures 5 - 7

 7 Figures 5 -7Hypaeus olympeae sp. nov. left male palp; 5. retrolateral view; 6. Same ventral view; 7. Same dorsal view showing tibial apophysis, scale 0.5mm.

  Figures 11 -13

  Hypaeus olympeae sp. nov. left male palp drawnings; 11. retrolateral view; 12. same, dorsal view showing tibial apophysis; 13 same, ventral view, scale 0.5mm.

Figures

  Figures 14 -16

  Hypaeus olympeae sp. nov. pictures of living specimens; 17. male holotype;18. female paratype.

  Hypaeus[START_REF] Simon | Etudes arachnologiques. 30e Mémoire. XLVII. Descriptions d'espèces nouvelles de la famille des Attidae[END_REF] type species Acragas taczanowskii[START_REF] Mello-Leitão | Contribuição ao conhecimento da fauna araneológica das Guianas[END_REF].Hypaeus olympeae Courtial & Picard sp. nov. Hypaeus olympeae sp. nov. are similar to those of H. miles Simon, H. femoratus Araújo & Ruiz and H. terraemediae Araújo & Ruiz for having dilated femora (Fig 4). However, H. olympeae sp. nov. has wider rounded TA, which is small and pointed in aforementioned species. Females of H. olympaeae sp. nov. are similar to those of H. femoratus and H. terraemediae (the female of H. miles is unknown). Digitiform glands curve from the center of the epigyne to the antero-lateral portion. Females of H. olympeae sp. nov. are slightly similar in shape to those of H. femoratus and H. terremaediae but the COs openings are less curved and space further apart them is bigger.

	Figs 1-18
	Diagnosis
	Males of
	Class Arachnida Cuvier, 1812
	Order Araneae Clerck, 1757
	Family Salticidae Blackwall, 1841
	Subfamily Salticinae Blackwall, 1841
	Tribe Amycini F.O Pickard-Cambridge, 1900
	Genus Hypaeus Simon, 1900

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(scale bar = 0.01 substitutions). See Table1for complete voucher information.

Acknowledgements

The authors thank Marguerite Delaval (Office National des Forêts, ONF) and ONF for funding the expedition to the Trinité National Nature Reserve; Christine Rollard and Christophe Hervé (Museum National d'Histoire Naturelle -Paris) for allowing access to the bibliography of the Arthropod laboratory and permitting the consultation of Caporiacco and Simon's collection; and Alain Canard, Boris Leroy, Frédéric Ysnel and Vincent Vedel for field support; Manuel Plantegenest, Yoann Navasse and El Aziz Djoudi (Agrocampus Ouest -Rennes) for laboratory access and technical support.

Funding

This work was supported by the Office National des Forêts (ONF).

Disclosure statement

No potential conflict of interest was reported by the authors.

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