Height and weight measurement were obtained from 164 healthy women of reproductive age attending gynaecological outpatient clinics in NHS Lothian. Written consent was obtained from all participants and a favourable ethical opinion granted from Lothian Research Ethics Committee (REC 15/WS/0212, REC 10/S1402/59, REC 07/S1103/29, REC 1994/6/17). All women reported regular menstrual cycles (21–35 days). The women were not using hormonal contraceptives and had no exogenous hormone exposure for 2 months prior to participation. In this study, 121 women returned a fully completed pictorial-based assessment chart (PBAC; Fig. 1) (Table 1). The PBAC contains pictorial representation of graded staining from slight to severe, across different absorbencies of menstrual towels and tampons. This is a validated technique and correlates with objective menstrual blood loss measurements obtained using the alkaline haematin method (Wyatt et al. 2001). Participants were asked to complete the PBAC each time they changed their menstrual pad/tampon over one menses. A scoring system for the PBAC was based on previous studies to give an estimated MBL in millilitres (Higham et al. 1990, Wyatt et al. 2001).

Participant recruitment. PBAC, pictorial-based assessment chart.

Summary of characteristics of 121 participants completing the pictorial-based assessment chart (PBAC) as mean (range) or number (%).

*Measurements only in women with fibroids present.

Endometrial biopsies (n  = 28) were collected during the late secretory or menstrual phase with a suction curette (Pipelle, Laboratorie CCD, Paris, France) from a subset of participants without fibroids >3 cm or symptoms of endometriosis (Table 1). Twenty women had a BMI <30 and 8 a BMI >30. Tissue was divided and (i) placed in RNA later, RNA stabilization solution (Ambion (Europe) Ltd., Warrington, UK), (ii) fixed in 4% neutral buffered formalin for wax embedding. Biopsies were confirmed as late secretory/menstrual by (i) histological dating (criteria of Noyes et al. 1950), (ii) reported last menstrual period and (iii) serum progesterone and oestradiol concentrations at the time of biopsy (Table 2).

Serum hormone levels in women providing endometrial biopsies.

Menstrual blood loss (MBL) was objectively measured in the women providing endometrial tissue using a modified alkaline haematin method (Hallberg & Nilsson 1964, Maybin et al. 2017) and heavy menstrual bleeding (HMB) defined as a blood loss of >80 mL per cycle. Women were given the same brand of menstrual products (Tampax® tampons/Always® towels, Proctor & Gamble, UK) with verbal and written instruction on collection. The technique was validated in our laboratory using a known volume of whole blood applied to menstrual products. The proportion of women with HMB in those with a BMI <30 and >30 who provided endometrial biopsies was 60% and 50%, respectively.