Endometrial receptivity and conception outcome among women with light menstrual bleeding of unidentified etiology

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1 Received: 31 January 2017 Revised: 4 August 2017 Accepted: 29 September 2017 First published online: 23 October 2017 DOI: /ijgo CLINICAL ARTICLE Gynecology Endometrial receptivity and conception outcome among women with light menstrual bleeding of unidentified etiology Yueqing Gao 1 Xiangli Hong 2 Zhewei Wang 1, * Ying Zhu 1 1 Department of Gynecology, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, China 2 Department of Ultrasound, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, China *Correspondence Zhewei Wang, 536 Changle Rd, Shanghai , China. wangzhewei99@163.com Abstract Objective: To investigate endometrial receptivity (ER), conception outcome, and other factors among women with light menstrual bleeding of unidentified etiology. Methods: A prospective study was undertaken at a center in China. Between January 1, 2011, and December 31, 2014, women meeting the inclusion criteria (aged years, without any history of pregnancy, desire to conceive, follicle- stimulating hormone <10 IU on second or third day of cycle, and regular menstrual periods) were enrolled. Participants with a menstrual blood volume of 5 20 ml formed the study group; others were in the control group. ER measures were obtained by transvaginal Doppler ultrasonography. Serum estradiol and progesterone levels were measured and items of prethrombotic state were screened in mid- luteal phase. Participants were followed up for 1 year to establish conception outcome. Results: The ER (Salle) score was significantly lower in the study group (n=110) than in the control group (n=330; 9.41 ± 3.02 vs ± 2.63, P<0.001). The natural rate of conception within 1 year was lower (60.0% [63/105] vs 72.1% [230/319], P=0.020), and the spontaneous abortion rate in early pregnancy was higher (34.4% [21/61] vs 18.1% [41/227], P=0.006) in the study group than in the control group. Conclusion: Women with light menstrual bleeding of unidentified etiology had an increased prevalence of low ER and poor conception outcome. KEYWORDS Endometrial receptivity; Infertility; Menstrual disorder; Prethrombotic state; Spontaneous abortion 1 INTRODUCTION The volume of menstrual blood ranges from 20 to 60 ml for most Chinese women. 1 Traditionally, doctors have focused on treatment for menorrhagia (menstrual blood volume >80 ml) and hypomenorrhea (menstrual blood volume <5 ml). Few studies have examined the effects of menstrual blood volumes of 5 20 ml. To our knowledge, there is no medical term for this condition, which we refer to as light menstrual bleeding. Furthermore, many cases of light menstrual bleeding cannot be attributed to a specific cause such as endocrine dysfunction, endometrial disease, or any other organic disease 2 ; we refer to this condition as light menstrual bleeding of unidentified etiology. Much recent research has focused on endometrial receptivity (ER). 3 Changes in endometrial thickness, luminal epithelium, glandular epithelium, stromal development, and endometrial blood supply make the endometrium receptive to embryonic implantation. These changes are regulated by a unique sequence of factors that occurs at a specific timepoint. ER is optimal from 6 or 7 days after ovulation to about 4 days into the luteal phase a period called the window of implantation. 4 This is the best time to evaluate ER. ER can be assessed using molecular markers. 5 However, this method usually involves invasive procedures and is unacceptable to women who wish to become pregnant in the current cycle. Highresolution transvaginal Doppler ultrasonography is now being widely Int J Gynecol Obstet 2018; 140: wileyonlinelibrary.com/journal/ijgo 2017 International Federation of 37 Gynecology and Obstetrics

2 38 Gao ET AL. used to assess ER and is gaining increasing attention for its advantages of non- invasiveness, real- time monitoring, and predictability. 6 For example, Yuri et al. 6 used ultrasonography to assess ER among women experiencing infertility, showing that there was higher impedance in the uterine artery, lower endometrial flow, and lower endometrial width than among women with proven fertility. Others have proposed that endometrial thickness and pattern, and endometrial subendometrial blood flows, as measured by ultrasonography scanning, could be related to ER. 7,8 It is possible that light menstrual bleeding of unidentified etiology could be a manifestation of poor endometrial subendometrial blood flow and poor ER, and that affected women might have difficulty conceiving. The aim of the present study was therefore to investigate differences in ER and physiological indices that could influence ER, including luteal function and prethrombotic state (PTS), between women with and without light menstrual bleeding of unidentified etiology. An additional aim was to establish conception outcomes. 2 MATERIALS AND METHODS The prospective study was undertaken at the gynecologic clinic in Shanghai First Maternity and Infant Hospital, Shanghai, China, between January 1, 2011, and March 31, Between January 1, 2011, and December 31, 2014, women attending the clinic were invited to participate in the study if they were aged years, had never been pregnant, were using contraception but intended to become pregnant, attended the clinic to seek a pre- pregnancy checkup, had a follicle- stimulating hormone level of less than 10 IU on the second or third day of the menstrual cycle, and had regular menstrual periods (cycle of days) with ovulation (as determined by selfreported basic body temperature). The results of the semen tests of participants spouses were normal. The exclusion criteria were endocrine disease (e.g. diabetes or thyroid disorder), systemic immune disorders, serious diseases of vital organs, tuberculosis, genital tract malformation, adenomyosis or uterine fibroids (with uterine volume >2 months of pregnancy), intrauterine adhesions or other mucosa lesion, blood system disease, or previous intrauterine operation. This information was verified by the patient s medical history, laboratory tests, and imaging examinations. The study was approved by the ethics committee of the study hospital. Informed consent was obtained from all participants. Menstrual blood volume was estimated by the participants using a pictorial blood loss assessment chart 9 for at least three cycles. Women whose mean menstrual blood volume ranged from 5 to 20 ml were included in the study group, whereas those with menstrual blood volume ranging from 20 to 80 ml were included in the control group. Women with a menstrual blood volume of greater than 80 ml were excluded. Before enrollment, potential participants were told that the study would be explorative and there would be no relative intervention guide, but the conclusion of the study might help participants with problems in conceiving. If a participant were to seek any treatment on infertility during the follow- up period, she would be regarded as a drop- out. Participants would be followed up every 3 months, and were asked to provide a cell phone number (verified with the hospital) and home phone number. Information on demographic status and history of menstruation, disease, and surgery was collected from participants. Ovarian reserve function (determined as the level of serum follicle- stimulating hormone) was tested on the second or third day of the menstrual cycle. Ovulation was monitored by transvaginal ultrasonography (including endometrial morphology and thickness on ovulation day) every 2 days beginning on days 5 10 of the first menstrual cycle after enrollment until the follicular diameter reached 15 mm. After the follicular diameter reached 15 mm, transvaginal ultrasonography was performed every day until the follicle was discharged. At 7 9 days after ovulation (during the mid- luteal phase), other ER variables (myometrial structure, pulsatility index [PI], early diastolic notch, end- diastolic blood flow of the uterine artery, and endometrial subendometrial blood flows) were observed by transvaginal ultrasonography. D- dimer and plasma fibrinogen levels, ADP- induced platelet aggregation (ADP- Ag), whole- blood viscosity, and prothrombin time were also measured 7 9 days after ovulation. To improve the accuracy of detecting luteal function, estradiol and progesterone levels were measured three times between the fifth and ninth day after ovulation, and the mean value was determined. 10 In the next menstrual cycle, the same blood indicators were measured again and the results were averaged. There are no global evaluation criteria for PTS; for the present study, therefore, the following comprehensive items for screening PTS in China were used 11,12 : level of D- dimer (0.55 mg/l fibrinogenequivalent units) or maximal intensity of ADP- Ag exceeding upper limit of normal range for the Chinese population (77.8%); abnormally high whole- blood viscosity on a shear rate of 3/s, 30/s, or 200/s; prothrombin time less than 9 seconds; or plasma fibrinogen more than 4 g/l. A Voluson730 expert color Doppler instrument (General Electric Company, New York, NY, USA) was used for ultrasonography. The ascending branch of the uterine artery was detected on two sides in a horizontal transverse section of the internal cervix after signal adjustment. The sampling space was 2 mm, and the sampling line was made consistent with the flow direction (i.e. angle with direction of blood flow <60 ). In six continuous and stable cardiac cycles, PI, early diastolic notch, and end- diastolic blood flow of the uterine artery were measured. Endometrial subendometrial blood flows were defined as small- vessel branches entering the inner and inferior halo observed on the screen. Endometrial morphology was classified by Dickey typing 13 : type A was indicated by three strong linear echo signals in the outer layer and midline, and weak echo in the endometrium; type B by three weak linear echo signals and unclear midline echo; and type C by homogeneous strong echo with no midline echo. ER was evaluated mainly by the Salle scale, comprising endometrial thickness, endometrial morphology, myometrial structure, PI, early diastolic notch, and end- diastolic blood flow of the uterine artery, and endometrial subendometrial blood flows. The maximum score of the scale is 20, and higher scores mean higher ER. 14

3 Gao ET AL. 39 Participants were subsequently followed up by telephone once every 3 months to establish conception status. The follow- up ended after 1 year if the participant had not become pregnant or until 13 weeks of pregnancy if conception had been achieved. Early pregnancy was confirmed by serum or urine human chorionic gonadotropin level. Early pregnancy outcomes were assessed by ultrasonography at weeks. Data analyses were performed with SPSS version 24.0 (IBM, Armonk, NY, USA). Differences between the study and control groups were assessed by χ 2 test or continuity adjusted χ 2 test for categorical variables, and t test for continuous variables. For all tests, the 95% confidence interval was calculated and P<0.05 was considered statistically significant. 3 RESULTS During the enrollment period, 462 women met the inclusion criteria: 114 had a menstrual blood volume of 5 20 ml and 348 had a menstrual blood volume of ml. Overall, 22 women refused to sign the informed consent form. Ultimately, 110 women were enrolled in the study group, and 330 women were enrolled in the control group. Mean age was ± 3.87 years in the study group and ± 3.83 years in the control group (t=1.662, P=0.097). There were no significant differences in ovarian reserve function or maximum follicle size before ovulation between the two groups (Table 1). The percentage of women with abnormally high ADP- Ag, D- dimer levels, and high whole- blood viscosity in mid- luteal phase was significantly higher in the study group than in the control group (all P<0.05) (Table 1). Additionally, the percentage of women with an abnormality in the PTS screening items was significantly higher in the study group (χ 2 =6.428, P=0.011) (Table 1). Although the levels of estradiol and progesterone were significantly lower in the study group (both P<0.001), the difference in rate of poor luteal function (<15 ng/ml) between the two groups was not significant (Table 1). The proportion of women with type A endometrium was significantly lower in the study group than in the control group (χ 2 =22.960, P<0.001) (Table 2). Endometrial thickness was also significantly lower (t=3.886, P<0.001) (Table 2). There was no significant difference in the myometrial structure, PI, or early diastolic notch or end- diastolic blood flow of the uterine artery between the two groups, but the endometrial subendometrial blood flow was significantly lower in the study group (t=20.154, P<0.001) (Table 2). Overall, the Salle score was significantly lower in the study group than in the control group (t=20.830, P<0.001) (Table 2). Sixteen women were lost to follow- up or withdrew (5 in the study group, 11 in the control group). Among the remaining women, the rate of spontaneous conception within 1 year was significantly lower in the study group than in the control group (χ 2 =5.418, P=0.020) (Table 3). Excluding five women with ectopic pregnancies, the rate of spontaneous abortion in early pregnancy was significantly higher in the study group than in the control group (χ 2 =7.622, P=0.006) (Table 3). TABLE 1 Ovarian function, blood coagulation, and endocrine and antibody levels in the mid- luteal phase. a Follicle- stimulating hormone, IU/L Maximum follicle diameter before ovulation, mm 4 DISCUSSION In the present study, evaluation of ER showed that the frequency of type A endometrium, endometrial thickness, and endometrial subendometrial blood flow were all lower in the study group than in the control group. Overall, the ER score was significantly lower among women with light menstrual bleeding of unidentified etiology than among control women. (n=110) (n=330) 6.19 ± ± ± ± Prothrombin time <9 s 8 (7.3) 12 (3.6) Fibrinogen >4 g/l 9 (8.2) 13 (3.9) Maximal intensity of ADP- induced platelet aggregation >77.8% 10 (9.1) 14 (4.2) D- dimer >0.55 mg/l FEU 11 (10.0) 11 (3.3) Abnormally high whole- blood viscosity Abnormality in PTS screening items 10 (9.1) 13 (3.9) (25.5) 49 (14.8) Estradiol, pg/ml ± ± <0.001 Progesterone, ng/ml ± ± 5.27 <0.001 Poor luteal function (<15 ng/ml) 17 (15.5) 31 (9.4) Abbreviations: FEU, fibrinogen- equivalent units; PTS, pre- thrombotic state. a Values are given as mean ± SD or number (percentage), unless stated otherwise. TABLE 2 Endometrial receptivity in the mid- luteal phase. a Type A endometrium in ovulation Endometrial thickness on ovulation day, mm (n=110) (n=330) 35 (31.8) 192 (58.2) < ± ± 2.32 <0.001 Uneven myometrial structure 21 (19.1) 69 (20.9) Pulsatility index of uterine artery >3 Diastolic notch or deficiency of end diastolic blood flow Endometrial subendometrial blood flow, counts of vessels 15 (13.6) 26 (7.9) (4.5) 6 (1.8) ± ± 4.30 <0.001 Salle score 9.41 ± ± 2.63 <0.001 a Values are given as number (percentage) or mean ± SD, unless indicated otherwise.

4 40 Gao ET AL. TABLE 3 Conception and early pregnancy outcomes. a Natural conception within 1 y Many studies have focused on the relationship between ER and infertility. It has been reported that the fertility rate reaches a peak for an endometrial thickness of 9 12 mm during the ovulatory period. 6,8 Additionally, type A endometrium on the day of human chorionic gonadotropin injection and oocyte retrieval has been associated with a significantly higher rate of pregnancy during infertility treatment. 15 Usually, the PI of the uterine artery, early diastolic notch, and end- diastolic blood flow are used to evaluate ER; however, these measures reflect the blood flow in the whole uterus. In fact, the blood flow surrounding the implantation site is of greater importance for embryo implantation. 16 A reduction in endometrial vascularity has been observed in women with unexplained infertility. 17 Studies using transvaginal color Doppler showed that good subendometrial blood flow distribution and sufficient blood flow in uterine vessels were positively correlated with positive pregnancy outcome. 17 In short, in the present study, the observed differences relevant to ER between the two groups indicate that light menstrual bleeding could be a marker of reduced ER, and that women with light menstrual bleeding of unidentified etiology could be predisposed to infertility and poor pregnancy outcomes. Although the present study found that estradiol and progesterone levels in the mid- luteal phase were statistically lower in the study group, the rate of poor luteal function (<15 ng/ml) was not significantly higher in the study group than in the control group. In terms of estradiol level in the mid- luteal phase, a normal range has not been established. Therefore, although lower estradiol levels were observed in the study group, the data are insufficient to indicate poor luteal function in women with light menstrual bleeding of unidentified etiology. Recently, several factors have been reported to be related to ER, including sex hormones and their receptors, angiogenesis factors, and natural killer cells in the endometrium. 18,19 PTS is the prophase of thrombotic disease and can cause microthrombosis at the site of the placenta, reduce endometrial microcirculation, and contribute to embryo damage in pregnancy if a high coagulation state occurs owing to the elevation of hormones in pregnancy. 20 PTS has also been related to poor ER. Low- dose aspirin can reduce ADP- Ag and improve endometrial microcirculation and ER. 21 Additionally, women with unexplained recurrent pregnancy loss were previously found to have increased ADP- Ag, and aspirin and low molecular weight heparin maintained a lower thrombotic state and led to a high rate of live birth for these women. 22 (n=105) (n=319) 63 (60.0) 230 (72.1) Spontaneous abortion in 21 (34.4) 41 (18.1) early pregnancy b a Values are given as number (percentage) unless indicated otherwise. b Among women who achieved pregnancy but excluding 5 women with ectopic pregnancy (2 in the study group and 3 in the control group). In Chinese traditional medicine, PTS is called blood stasis syndrome, and affected women have slow blood flow and decreased menstrual bleeding. Therefore, an investigation of changes in PTS might have both practical significance and clinical value. A previous study 23 found that a combined increase in fibrinogen and D- dimer levels might indicate the existence of PTS. Because blood viscosity was found to be significantly increased in a group of patients with small artery occlusion, 24 and ADP- induced platelet aggregation function is a predictive factor of poor coronary events, 25 these indices were included among the present items for comprehensive screening of PTS. The present study suggested that the percentage of abnormalities in these comprehensive screening items was statistically higher in the study group than in the control group. It is possible that these abnormalities could indicate the presence of PTS, which might be the underlying cause of their poor ER. To identify other reasons for poor ER among these women, further study is needed. The follow- up results showed that the rate of spontaneous conception within 1 year was significantly lower in the study group than in the control group, and the spontaneous abortion rate in early pregnancy was significantly higher. This supports the hypothesis that light menstrual bleeding of unidentified etiology is a manifestation of poor ER and leads to poor conception outcomes. It is important to review the limitations of the present study. First, estradiol levels were not measured during the late follicular phase. Second, owing to the limitation of research conditions, specific test items of PTS were not screened, and middle and late pregnancy outcomes of the women were not followed. Third, in China, hysterosalpingography is available only for women with infertility diagnosed by a clinician, and owing to a lack of funding, this test was not given to the study participants. These three limitations might have led to bias in the results. Additionally, ER was assessed only by physical appearance and not by biochemical markers because noninvasive and relatively cheaper Doppler is more acceptable and convenient among patients. In conclusion, the present study found that women with light menstrual bleeding of unidentified etiology have an increased frequency of poor ER and poor conception outcomes. For these women, especially those diagnosed with infertility or with a history of spontaneous abortion, an assessment of ER and early intervention might improve their conception outcome. AUTHOR CONTRIBUTIONS YG, XH, and ZW participated in research design. All authors performed the experiments, and wrote and revised the manuscript. YG and ZW performed data analysis. ACKNOWLEDGMENTS Lei Wang and Hua Zhang provided technical support. CONFLICTS OF INTEREST The authors have no conflicts of interest.

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