Urinary LH Research Articles

Utility of First-Morning-Voided Urinary Total Luteinizing Hormone in Detecting the Onset of Central Puberty

Introduction: Early morning basal serum luteinizing hormone (S-LH) ≥0.3 IU/L is a specific marker for the onset of central puberty. In this study, we aimed to investigate the sensitivity and specificity of the first-morning-voided (FMV) total urinary LH (U-LH) to replace this marker. Methods: We re-analyzed our previously published data set of 297 children (145 boys and 152 girls, aged 5–15 years, across Tanner stages 1 through 5) using receiver operating characteristic (ROC) analysis and determined cutoff values for FMV total U-LH in predicting early morning S-LH concentration at or above 0.3 IU/L. We also determined S-LH and serum follicle-stimulating hormone (S-FSH) concentrations in girls at different stages of sexual maturation. Results: ROC analysis showed that FMV total U-LH levels of 0.60 and 0.63 IU/L in girls and boys, respectively, predicted early morning S-LH levels of 0.3 IU/L or higher with 97.4% sensitivity and 90.6% specificity. Higher cutoff levels for U-LH (0.78 IU/L for boys and 0.79 IU/L for girls) yielded 94.7% specificity at the expense of a relatively lower level of sensitivity (94.1%). The areas under the curve were 0.98 in boys and 0.99 in girls, respectively. Additionally, the increase in FMV total U-LH (or S-LH) levels identified the activation of central pubertal development at the mean age of 10.3 (10.3) in boys and 10.5 (10.6) in girls. The S-FSH concentrations of the six biochemically prepubertal girls with thelarche, ranging between 2.3 and 2.7 IU/L, were significantly higher than those measured in biochemically and clinically prepubertal girls of the same 10–12-year-old age group and significantly lower than those measured in both biochemically and clinically pubertal girls (p = 0.039 and p = 0.018, respectively). Conclusions: A FMV total U-LH concentration of 0.6 IU/L or above reliably reflects pubertal morning S-LH levels and is effective in detecting the onset of central puberty, which occurs at similar ages in both sexes. Concurrent S-FSH or noninvasive FMV U-FSH determinations may be useful in the differential diagnosis of isolated thelarche.

The assessment of urinary sexual hormones within minipuberty and correlations with anthropometrics in a cohort of healthy term children.

Minipuberty follows different trends in boys and girls. Aim of our study was to explore timing and dynamics of minipuberty in healthy infants, analyzing urinary levels of sexual hormones. Moreover, we analyzed the association among HPG axis activity and linear growth, ano-genital distances (AGDs) in both sexes, and penile length in males. Longitudinal cohort study in healthy term infants from birth to 6 months of life. Clinical evaluation (anthropometrics and AGDs) and urine sampling were performed at 0 (T0), 3 (T3), and 6 (T6) months. Urine samples were analyzed for gonadotropins and sex hormones. 165 participants were involved. The growth trend of our population was regular, as were the AGDs. Urinary hormones were correlated each other's. Specifically, in boys, the correlation coefficient between urinary FSH (uFSH) and urinary LH (uLH) decreased from T0 to T6, while between urinary Testosterone (uT) and uFSH increased. In girls, correlations between uFSH and urinary Estradiol (uE) were observed at each time point. Notably, several correlations between hormones and anthropometrics and AGDs were found; the most interesting correlation was found in males within uLH and PL (at T0 ρ: 0.323, p < 0.05 and at T3 ρ: 0.371, p < 0.01), whereas in females uFSH at T0 showed negative correlations with both length and body weight percentile at T3 (ρ: -0.505, p < 0.01 and ρ: -0.478, p < 0.01, respectively). Urinary matrix has proved to be a valuable, practical, non-invasive and cheaper method for sexual hormone assessment.

Evaluation of ovulation by urinary LH surge kits versus transvaginal sonography

The study was conducted to evaluate the efficacy of urinary LH surge kits and transvaginal ultrasonography (TVS) to detect ovulation in induced cycles and to compare the ovulation rates by both methods. Prospective observational cohort study on 144 women with anovulatory infertility aged 18-35 years, fulfilling the inclusion criteria were given letrozole for ovulation induction. All were randomly divided into two groups. Group 1 women were asked to check ovulation by urinary LH surge kits and group 2 women were called for follicle monitoring by TVS. Letrozole has no negative effect on the endometrium; the induced cycle has a larger diameter of follicle (median: 22 mm). In the induced cycle ovulation occurs later compared to normal cycle (D-16) and half of the women had a BMI more than the recommended WHO criteria (average was 25.28 kg/m2). Number of letrozole cycles (p=0.2642), dose requirement (p=0.0812) and pregnancy rates (10.26% versus 18.19%) were comparable in both groups. TVS is objective, accurate, and thus a standard modality for ovulation detection. LH surge kit is subjective, having more chances of error but can be used as a good alternative in certain settings like women from the remote area, and women having fear of invasive modality.

P-315 Microbiota in endometriosis: culturomics analysis in receptive-phase endometrium

Abstract Study question Does the endometrium possess alive, culturable bacteria, and are there any microbes associated with endometriosis? Summary answer Thirty-five cultured bacterial species were identified in the receptive-phase endometrium, and an endometriosis-specific bacterial profile was detected. What is known already Endometriosis is a chronic inflammatory disease and one of the most prevalent female reproductive disorders, but its etiology remains unknown. Recently, significant attention has been directed towards studying the endometrial microbiome and its potential correlation with endometriosis. These analyses typically employ 16S rRNA gene sequencing, which detects bacterial genera but does not distinguish whether the bacteria are alive and hasn’t provided conclusive results regarding endometrial microbiome composition. Our aim is to develop a culture-based method for identifying live microbes in the endometrium and identifying bacterial species associated with endometriosis. Study design, size, duration This pilot cross-sectional study recruited twenty women from couples prior to undergoing infertility treatment at the Reproductive Unit of Universitary Hospital Virgen de las Nieves (Granada) between 2020 and 2022. Ten women were diagnosed with active endometriosis (no treatment), while ten women had male factor infertility, serving as the control group in our study. Participants/materials, setting, methods Endometrial biopsies were obtained during the mid-secretory phase, 7-9 days after ovulation (assessed with a urinary LH test), using an endometrial curette device. The biopsies were snap-frozen and stored at -80 °C until analysis. Microbiota cultures were conducted for up to 30 days using nine different media under aerobic, microaerobic, and anaerobic conditions. Matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS) was utilized for bacterial isolate identification. Main results and the role of chance In total, 19 out of 20 patients (95%) tested positive for one or more microbes, with only 1 patient (5%) showing no microbial growth. We detected thirty-five bacterial species in these mid-secretory endometrial samples. Among them, fourteen bacterial species were common in both endometriosis and control samples, while five species—Bifidobacterium bifidum, Bifidobacterium breve, Cutibacterium avidum, Lactobacillus vaginalis, and Staphylococcus warneri—were exclusively cultured from endometriosis samples. Conversely, healthy samples harbored fourteen exclusive species, including Actinomyces neuii, Corynebacterium amycolatum, Enterococcus faecalis, and Finegoldia magna, among others. Control endometrial samples, altogether, possessed significantly more bacterial species than endometriosis samples (30 vs. 19 microbial species). These findings are consistent with the initial culturomics analyses of endometrial microbiota, highlighting the method’s efficacy in identifying isolates at the species level. Notably, species such as Actinotignum schaalii, Bifidobacterium dentium, Atopobium vaginae, and Rothia dentocariosa were identified in our samples, marking the first description of these species in endometrial culturomics studies. Among all participants, the bacteria with the highest colony forming units were Cutibacterium acnes, Lactobacillus jensenii, Lactobacillus crispatus, and Lactobacillus iners. Our study confirms the presence of the endometrial microbiota and suggests microbial dysregulation in endometriosis. Limitations, reasons for caution This is a pilot study with a small sample size, requiring further investigation with a larger cohort. The developed protocol analyzed frozen samples, which may have failed to detect some of the fragile microbial species. Wider implications of the findings The identification of live culturable bacteria through culturomics in endometrial biopsies offers a reliable method for detecting endometritis-associated bacteria. Utilizing culturomics for endometrial microbiota testing shows promise in uncovering specific bacteria associated with gynecological disorders like endometriosis, and it enhances our understanding of potential pathological processes. Trial registration number not applicable

P-436 Uterine NK cells underexpress KIR2DL1/S1 and LILRB1 receptors recognizing HLA-C2 and HLA-G on extravillous trophoblasts in reproductive failure

Abstract Study question Is there a difference in frequency, receptor expression and cytokine production of uterine NK cells (uNK) subpopulations in women with reproductive failure compared to controls? Summary answer Reproductive failure is associated with global underexpression of uNK receptors interacting with extravillous trophoblasts (EVT) that may result in defective implantation and placentation. What is known already A significant proportion of infertility, recurrent miscarriage and recurrent implantation failure are unexplained. This may be caused by immunological dysfunction at the maternal-fetal interface resulting in defective implantation and placentation. uNK is thought to play an important role in early pregnancy by cross-talking with EVT via KIR/HLA-C, LILRB1/HLA-G and CD94/HLA-E interactions to regulate EVT invasion and spiral arteriole remodeling in the decidua. Dysregulation of uNK function may cause reproductive failure; however it is unclear whether NK cell over- or under-activation is the pathology. Previous studies have been conducted on uNK obtained after miscarriage, which may be confounded by inflammatory processes. Study design, size, duration Here, we conducted a cross-sectional study to assess frequency, phenotype and function of matched pNK and uNK during the window of implantation in 16 women with reproductive failure compared to 11 controls. Women with reproductive failure cohort included unexplained subfertility (9), recurrent miscarriage (4) and recurrent implantation failure (3). The women were subsequently followed up after IVF and comparison made between women who did and did not fall pregnant. Participants/materials, setting, methods Matched peripheral blood and endometrial tissue were obtained from participants during the mid-secretory phase. Samples were accurately timed for 7-9 days after ovulation by urine LH test or histological dating. Flow cytometry was used to assess the frequency, phenotype and function. Phenotype was measured by KIR, LILRB1 and CD94 and receptor expression. Function was measured by degranulation (anti-CD107a internalization) and cytokine production (TNF-α, GM-CSF, IFN-γ and IL-8), with and without stimulation by PMA and ionomycin. Main results and the role of chance The expression of KIR2DL1/S1 and LILRB1 were significantly lower in reproductive failure group for both uNK (total uNK, uNK2 and 3) and pNK. The common finding in both uNK and pNK suggests that this is not tissue specific and may have an immunogenetic aetiology. We have previously found that degranulation peaks during secretory phase and in first trimester pregnancy. TNF-α production was also found to peak in secretory phase. Here, we found that degranulation activity of total uNK, as well as TNF-α production all uNK subsets was significantly lower in reproductive failure compared to control group. However, no significant difference was found in women in the reproductive failure cohort who fell pregnant compared to women who did not fall pregnant or miscarried after IVF. Contrary to findings from previous studies, we have not found any difference in uNK or pNK frequencies in any of the groups that were examined. Taken together, our findings suggest that reproductive failure is associated with global underexpression of receptors important for interaction with HLA-C and HLA-G on EVT, resulting in reduced uNK activation. This in turn may lead to dysregulation of implantation and placenta formation in early stage of pregnancy. Limitations, reasons for caution KIR2DL1/S1 antibody used does not distinguish between KIR2DL1 and KIR2DS1 which exerts inhibitory and activating effects respectively. Larger cohort studies on both pNK and uNK that consider KIR2DL1 and KIR2DS1 separately is needed to confirm which of these receptors are specifically reduced in reproductive failure. Wider implications of the findings Overall, our findings suggest that reproductive failure is associated with global underexpression of NK cell receptors leading to impaired implantation and placentation. This is the first study to examine uNK subsets during window of implantation and will serve as a platform to focus on particular uNK subsets in future studies. Trial registration number Not applicable

Role of luteinizing hormone urinary levels in the diagnostic and therapeutic management of female central precocious puberty

BackgroundDiagnosing central precocious puberty (CPP) requires an integrated approach based on clinical, biochemical and instrumental data. The diagnostic gold standard is represented by GnRH (gonadotropin-releasing hormone) stimulation test. Some undoubted limitations of this procedure led the international scientific community to look for cheaper and less invasive alternative diagnostic methods, such as luteinizing hormone urinary levels (uLH) measurement. This study aims to define the reliability of urinary LH levels as a biomarker of pubertal development, both concerning the initial diagnostic management and the monitoring of patients with central precocious puberty undergoing therapy with GnRH analogues. Furthermore, the study plans to detect the potential association between LH peak serum (pLH) and urinary LH in patients undergoing diagnostic tests with GnRH and to identify a possible cut-off of uLH that may be suggestive of ensued successful hormonal stimulation.MethodsThe study includes 130 female patients with suspected precocious puberty or in follow-up during suppressive therapy. After the collection of the informed consent, the patients underwent clinical evaluation, auxological assessment, and hormone assays (basal levels of LH, FSH, and oestradiol; GnRH stimulating test in patients with suspected precocious puberty; urinary LH assay on the first-morning urine sample, collected after waking up).ResultsTwo uLH cut-off values have been identified: the first of 0.25 UI/L [C.I. 95% 0.23–0.27], able to distinguish between pubertal and pre-pubertal patients, the second of 0.45 UI/L [C.I. 95% 0,20 − 0,70] suggestive of occurred hormonal stimulation in patients with diagnosis of CPP at GnRH test. All 30 patients with CPP in follow-up during suppressive therapy presented uLH values ≤ 0.45 IU/L (pU < 0.05), and uLH collected in prepubertal group control.ConclusionsuLH assays on the first morning urine specimen could be considered a low-cost and minimally invasive tool for precocious puberty diagnosing and monitoring, making possible to be easily performed even by a general pediatrician. Thus, this could help referring only selected patients to pediatric endocrinologists. After an appropriate validation, this approach could reasonably reduce hospital attendance and costs of performing more invasive procedures, with a more significant emotional impact on the pediatric patient.

Effect of Menstrual Pain on Sleep Quality and Anaerobic Performance

This study aimed to examine the effect of menstrual pain on sleep quality and anaerobic performance in women and the relationship between menstrual symptoms, sleep quality, and anaerobic performance. The study consisted of 22 (20.64 ± 1.91 years) healthy young women. The participants were divided into two groups according to their verbal expressions as menstruating with pain (n=11) and menstruating without pain (n=11). The study used a calendar-based counting method reported by women and an ovulation prediction kit to determine urinary LH surge. After the menstrual cycle phases were determined, Wingate anaerobic performance test measurements were applied to the participants in three phases (late follicle, ovulation and mid-luteal phase). Besides, the Pittsburgh sleep quality index was used to determine women’s sleep quality, and the menstrual symptom scale was used to measure menstrual symptoms. Mann-Whitney U test, one of the non-parametric tests, was used in the data analysis. In statistical analysis, the level of significance was accepted as p &amp;lt; 0.05. Spearman correlation analysis was used to determine the relationship between menstrual pain, sleep quality and anaerobic performance. As a result, the average sleep quality of the participants was higher than the painless group. Menstrual symptom scale "pain symptoms", and "coping methods" scores and menstrual symptom scale total scores differed between the groups. The study found that the power drop score, one of the anaerobic performance scores, differed between the groups in the mid-luteal phase. As a result of the correlation test, menstrual pain and sleep quality were associated with anaerobic performance.

Follicular and Hormonal Changes after Estrous Synchronization in Bottlenose Dolphins.

To gain more knowledge about the influence of hormone regulation on follicle development, ovarian ultrasounds were performed, and urinary hormone profiles were determined in ovulating and non-ovulating female bottlenose dolphins (n = 15) following estrus synchronization with altrenogest. Ovarian ultrasounds were conducted daily, post-synchronization to describe follicular recruitment in relation to the endocrine profile. Follicle sizes were grouped into very small (VSM), small (SM), medium (MD) and large (LG). In ovulating females, two follicular waves were identified, and follicular deviation toward establishing a dominant follicle only occurred during the second wave. For non-ovulating females, only the first wave was observed. For all urinary hormones, the non-ovulating group presented significantly lower concentrations of follicle-stimulating hormone (uFSH), luteinizing hormone (uLH), estrone conjugates (uE1-C) and estriol (uE3) but similar progestagen and cortisol concentrations compared to the ovulating group. Concentrations of uE1-C and uE3 and the numbers of MD and LG follicles significantly (P < 0.05) increased, while uFSH concentrations significantly (P < 0.05) decreased as ovulation approached. Urinary LH significantly increased concurrently with increasing numbers of LG follicles and decreasing numbers of SM follicles. The characterization of follicular development and its relationship with hormone assessment complements our understanding of follicular recruitment post-synchronization in bottlenose dolphins and provides new information concerning differences between ovulating and non-ovulating females in response to an estrous synchronization protocol.Lay summaryMany species of fresh and saltwater dolphins and porpoises are facing increasing pressure for food and habitats due to human activities. One of the primary aspects that can be affected by such activities is reproduction. The bottlenose dolphin has been successfully bred in zoos and aquariums for at least 70 years, and they can be trained for several voluntary behaviors that enable medical examinations. Therefore, they represent a unique resource for research and understanding of normal dolphin reproduction. In this research, voluntary ultrasound exams and urine samples from 15 female dolphins were used to describe changes in their ovaries. The resulting descriptions and comparisons provide insight into the unique ovarian physiology of bottlenose dolphins and into the differences between animals after treatment. This information adds to the body of knowledge which one day may be used for developing advanced reproductive techniques to help preserve endangered species of dolphins.

P-366 Tumor necrosis factor-alpha and milk fat globule-epidermal growth factor 8: novel biomarkers to predict implantation failure and pregnancy loss

Abstract Study question Can implantation failure (IF) and pregnancy loss be predicted in serum prior to in vitro fertilization (IVF)? Summary answer Tumor necrosis factor (TNFα) and milk fat globule-epidermal growth factor 8 (MFG-E8) levels may serve as serum markers to predict IF and pregnancy loss. What is known already In a normal pregnancy, mediators such as TNFα are released creating a physiological inflammatory response. However, an exaggerated release of TNFα has been associated with IF and recurrent pregnancy loss (RPL). Recent studies demonstrated that TNFα up-regulates the expression of inflammatory factors such as MFG-E8. MFG-E8 is known to modulate implantation by acting at various levels of the trophoblast and endometrial compartments. Hence an overexpression of this protein may result in apoptosis, endometrial damage, and impaired implantation. Study design, size, duration This multicentric prospective controlled pilot clinical study was conducted from January 2016 to January 2020 and included 30 women in their natural cycle in which serum MFG-E8, TNFα, estradiol (E2), and progesterone (P4) levels were quantified in the early proliferative (cycle day 2) and secretory phases (urinary LH + 7 days). Additionally, an endometrial biopsy was obtained on urinary LH + 7 days for MFG-E8 and TNF α protein and gene expression analysis. Participants/materials, setting, methods Women ages 21-35y were recruited from 3 groups: fertile controls (C), unexplained IF (following 3 failed good quality embryo transfers), and RPL (at least 2 unexplained first trimester miscarriages). Patients with history of uterine surgery, abnormal uterine cavity (fibroids, endometrial polyps, adhesions, adenomyosis, and congenital uterine abnormalities), hydrosalpinx, diminished ovarian reserve, harboring chromosomal rearrangements, thrombophilia, or autoimmune diseases were excluded. Main results and the role of chance Ten women were included in each group. No statistical differences were found in age, BMI, AMH, baseline FSH, and baseline antral follicle count among cohorts. Mean serum E2 and P4 levels were similar among all groups in both the proliferative and secretory phases: E2 proliferative (C 69.19±26.64 pg/ml, IF 64.19±32.56 pg/ml, RPL proliferative 57.44±38.51; p = 0.55), E2 secretory (C 164.10±52.57 pg/ml, IF 172.57±121, RPL 173.81±.97.35; p = 0.25), P4 proliferative (C 0.45±0.15 ng/ml, IF 0.45±0.19 ng/ml, RPL 0.53±0.18 ng/ml; p = 0.85), P4 secretory (C 7.42±4.06 ng/ml, IF 7.8±4.56 ng/ml, RPL 8.05±4.38 ng/ml; p = 0.74). Mean serum TNFα levels were significantly higher in both, the proliferative and secretory phases for the RPL group (proliferative RPL 9.98±4.47 pg/ml, IF 4.73±2.56 pg/ml, C 3.42±1.01 pg/ml; p = 0.001 vs secretory RPL 8.67±4.45 pg/ml, C 3.35±0.94 pg/ml, IF 3.85±1.01 pg/ml; p = 0.03). Mean serum MFG-E8 levels were significantly higher in the IF group during the proliferative phase (IF 373±201 pg/ml, RPL 201±115 pg/ml, C 225.58±109.73pg/ml; p = 0.03), but not in the secretory phase (IF 237±101 pg/ml, RPL 189±116 pg/ml, C 199.41±112.43 pg/ml; p = 0.15). Endometrial MFG-E8 mRNA levels were significantly lower in the IF and RPL group compared to C (p = 0.03). TNFα mRNA levels were not statistically significant among groups (p = 0.12). Limitations, reasons for caution This is a pilot study to assess feasibility. Due to the small sample size, the effects of more subtle covariates would not have been detected. Future larger studies are warranted. Wider implications of the findings These novels differentially expressed serum and endometrial markers may provide information on the physiology of implantation and could generate the basis for non-invasive diagnostic tools and therapeutic use of MFG-E8/TNFα antagonists in women with IF and RPL. Trial registration number IIT-2014-100366

P-106 Total motile sperm count and oral ovulation induction protocols are not predictors of success in donor insemination cycles: Results from a large retrospective cohort study

Abstract Study question to investigate the effect of total motile sperm (TMS) count, and ovulation induction on clinical pregnancy rate in artificial insemination with donor (AID) cycles. Summary answer TMS count is not a predictor of sucess, and natural cycles are as effective as oral ovulation induction in donor inseminations What is known already There are inconsistent results regarding the effect of sperm parameters on success rates of artificial insemination with donor (AID). Besides, the use of gonadotropin stimulation for ovulation induction in this category of patients is questionable, and its effectiveness over natural cycles is not yet confirmed in the literature. Study design, size, duration Patients who underwent AID cycles at the university-affiliated fertility center-OVO clinic in Montreal, Canada between 2011 and 2015 were retrospectively selected. A total of 4333 AID cycles were performed on 1179 patients, resulting in 744 pregnancies. Participants/materials, setting, methods Cycles were divided into 8 groups based on TMS count: &amp;lt;0.5, [0.5-1[, [1-5[, [5-10[, [10-20[, [20-40[, [40-80[, and ≥80. A TMS of 10 to 20 million was selected as a reference level. Ovulation induction was divided into oral stimulation, combined oral and gonadotropin stimulation and gonadotropin-only stimulation, and compared to natural cycles. Regression analysis and a predictive model of clinical pregnancy in AID cycles were generated from patient demographic and cycle characteristics. Main results and the role of chance There was no significant difference in positive β-hCG result, clinical pregnancy, multiple pregnancy and miscarriage rates when comparing all ranges of TMS count to a reference of 10 to 20 million. When dividing patients based on the protocol for ovulation induction, clinical pregnancy rate was significantly higher in the gonadotropin-only stimulation group (OR 4.116,[1.379,12.287]) but not in other types of stimulation, as compared to natural cycles. hCG triggering resulted in a similar clinical pregnancy and miscarriage rates, but a higher multiple pregnancy rate when compared to urinary LH testing (7.7% versus 1.3%, p = 0.045). A multivariate logistic regression analysis for predictors of clinical pregnancy accounting for relevant demographic and cycle characteristics was conducted. No significant difference was noted in different ranges of TMS and the groups of ovulation induction. In this model, age was found to be a significant predictor. In particular, with every one-year increase in age, the odds of clinical pregnancy decreases by 6.4% (Adjusted OR 0.936; 95%CI [0.914, 0.958]). Limitations, reasons for caution In our study, cycles with TMS count below 1 million are limited. Thus, results should be viewed with caution in this group, without cycle cancellation, since clinical pregnancy can be achieved. Moreover, our results cannot be generalized on infertile couples given the characteristics of our population of interest. Wider implications of the findings Minimal or maximal cut-off values for TMS in AID cycles should not be used as indicators for cycle cancellation. Natural cycles are as successful as oral ovulation induction. hCG trigger, unless indicated, should not be used as it is associated with higher risk of multiple pregnancy without increasing clinical pregnancy. Trial registration number not applicable

The LH surge and ovulation re-visited: a systematic review and meta-analysis and implications for true natural cycle frozen thawed embryo transfer.

Efficient and safe embryo vitrification techniques have contributed to a marked worldwide increase in the use of elective frozen embryo transfer (FET). Pinpointing the day of ovulation, more commonly by documentation of the LH surge and less commonly by ultrasonography, is crucial for timing of FET in a true natural cycle (t-NC) to maximize the reproductive outcome. The definition of the onset of the LH surge should be standardized in t-NC FET cycles; however, a clear definition is lacking in the available literature. The first search question concerns the definition of the onset of the LH surge in a natural cycle. The second search question relates to the duration between the onset of the LH surge and ovulation. We searched PubMed, Web of Science and Cochrane Library databases for two search questions from inception until 31 August 2021. 'Luteinizing hormone'[MeSH] OR 'LH' AND 'surge' terms were used to identify eligible articles to answer the first question, whereas 'Luteinizing hormone'[MeSH] OR 'LH' AND 'surge' OR 'rise' AND 'ovulation'[MeSH] OR 'follicular rupture' OR 'follicular collapse' were the terms used regarding the second question. The included publications were all written in the English language, conducted in women of reproductive age with regular ovulatory cycles and in whom serial serum or urine LH measurement was performed. For the quality and risk of bias assessment of the included studies, the Strengthening the Reporting of Observational Studies in Epidemiology and modified Newcastle Ottawa Scale were used. A total of 10 and 8 studies were included for search Questions 1 and 2, respectively. Over the years, through different studies and set-ups, testing in either serum or urine, different definitions for the onset of the LH surge have been developed without a consensus. An increase in LH level varying from 1.8- to 6-fold above the baseline LH level was used in seven studies and an increase of at least two or three standard deviations above the mean of the preceding LH measurements was used in two studies. An LH level exceeding the 30% of the amplitude (peak-baseline LH level) of the LH surge was defined as the onset day by one study. A marked inter-personal variation in the time interval between the onset of the LH surge and ovulation was seen, ranging from 22 to 56 h. When meta-analysis was performed, the mean duration in hours between the onset of the LH surge and ovulation was 33.91 (95% CI = 30.79-37.03: six studies, 187 cycles). The definition of the onset of the LH surge should be precisely defined in future well-designed studies employing state-of-art laboratory and ultrasonographic equipment. The window of implantation in a natural cycle is still a black box, and future research is warranted to delineate the optimal interval to time the embryo transfer in t-NC FET cycles. Randomized controlled trials employing different precise endocrine and/or ultrasonographic criteria for timing of FET in a t-NC are urgently required.

Outcome of vitrified-warmed blastocyst transfer performed on days 5–7 after urine LH detection

Research questionWhat is the clinical importance of vitrified-warmed blastocyst transfer timing if performed on days 5, 6 and 7 after detecting the LH surge using urine tests? DesignBetween 2013 and 2019, 2080 vitrified-warmed blastocyst transfers in a true natural cycle were performed and later analysed at the Department of Reproductive Medicine, University Medical Centre Maribor, Slovenia. Urine LH tests were performed twice daily to monitor the onset of the LH surge. Vitrified-warmed blastocyst transfer (frozen embryo transfer [FET]) was performed on day 5 (group 1), 6 (group 2) or 7 (group 3) after the LH surge in 18%, 77% and 4% of cycles, respectively. The patient and cycle characteristics among the groups were compared using the Cochran–Mantel–Haenszel test and respective generalized linear mixed models. Propensity score matching was used to adjust for potential differences among the groups. ResultsThere were no statistically significant differences between groups 1, 2 and 3 in the cycle and patient characteristics, clinical pregnancy rate (38% versus 39% versus 31%), implantation rate (34% versus 36% versus 31%), miscarriage rate (7% versus 9% versus 7%) and delivery rate (31% versus 31% versus 24%). The day of FET after the LH surge detected using a urine test was not significantly associated with live births. ConclusionsThe results of the current study suggested that the vitrified-warmed blastocyst transfer could be scheduled on day 5, 6 or 7 after a positive LH urine test without having a significant impact on the clinical outcome.

Reproducibility and Refinement of Urinary LH in the Screening of Progressive Puberty in Girls.

First-voided urinary LH (FVU-LH) has been suggested as an alternative to GnRH stimulation test for detection of precocious puberty. To evaluate the reproducibility of FVU-LH, its correlation with basal and GnRH-stimulated gonadotropins, and its diagnostic value for differentiating progressive from nonprogressive puberty. Clinical and endocrine data were obtained from the medical records of 95 girls with suspected progressive puberty who underwent 2 consecutive FVU-LH tests. In 55 of these participants, GnRH stimulation test was performed close to the FVU-LH test. The reported cutoff levels of 5 IU/L and 1.16 IU/L for GnRH-stimulated LH and FVU-LH, respectively, were used as markers of progressive puberty, clinically defined as bone age advancement of ≥1 year and/or growth velocity SD score ≥2, in addition to thelarche. The 2 consecutive measurements of FVU-LH were highly correlated (r = 0.830; P < 0.001). The higher of the 2 results was better correlated with basal gonadotropins and GnRH-stimulated LH. Furthermore, it aligned better with the clinical outcome of girls with early thelarche, which supports the approach of double tests of FVU-LH to distinguish progressive from nonprogressive puberty. By comparison to GnRH-stimulated LH, the higher FVU-LH value had better sensitivity (68%), whereas peak LH had better specificity (91%) for the diagnosis of progressive puberty. Both tests had high positive predictive value and poor negative predictive value. The higher value of paired FVU-LH tests can be used to screen girls with suspected progressive puberty and can reduce the need for GnRH stimulation test.

Is home-based monitoring of ovulation to time frozen embryo transfer a cost-effective alternative for hospital-based monitoring of ovulation? Study protocol of the multicentre, non-inferiority Antarctica-2 randomised controlled trial.

STUDY QUESTIONThe objective of this trial is to compare the effectiveness and costs of true natural cycle (true NC-) frozen embryo transfer (FET) using urinary LH tests to modified NC-FET using repeated ultrasound monitoring and ovulation trigger to time FET in the NC. Secondary outcomes are the cancellation rates of FET (ovulation before hCG or no dominant follicle, no ovulation by LH urine test, poor embryo survival), pregnancy outcomes (miscarriage rate, clinical pregnancy rates, multiple ongoing pregnancy rates, live birth rates, costs) and neonatal outcomes (including gestational age, birthweight and sex, congenital abnormalities or diseases of babies born).WHAT IS KNOWN ALREADYFET is at the heart of modern IVF. To allow implantation of the thawed embryo, the endometrium must be prepared either by exogenous oestrogen and progesterone supplementation (artificial cycle (AC)-FET) or by using the NC to produce endogenous oestradiol before and progesterone after ovulation to time the transfer of the thawed embryo (NC-FET). During an NC-FET, women visit the hospital repeatedly and receive an ovulation trigger to time FET (i.e. modified (m)NC-FET or hospital-based monitoring). From the woman’s point of view, a more natural approach using home-based monitoring of the ovulation with LH urine tests to allow a natural ovulation to time FET may be desired (true NC-FET or home-based monitoring).STUDY DESIGN, SIZE, DURATIONThis is a multicentre, non-inferiority prospective randomised controlled trial design. Consenting women will undergo one FET cycle using either true NC-FET or mNC-FET based on randomisation.PARTICIPANTS/MATERIALS, SETTING, METHODSBased on our sample size calculation, the study group will consist of 1464 women between 18 and 45 years old who are scheduled for FET. Women with anovulatory cycles, women who need ovulation induction and women with a contra indication for pregnancy will be excluded. The primary outcome is ongoing pregnancy. Secondary outcomes are cancellation rates of FET, pregnancy outcomes (including miscarriage rate, clinical pregnancy, multiple pregnancy rate and live birth rate). Costs will be estimated by counting resource use and calculating unit prices.STUDY FUNDING/COMPETING INTEREST(S)The study received a grant from the Dutch Organisation for Health Research and Development (ZonMw 843002807; www.zonmw.nl). ZonMw has no role in the design of the study, collection, analysis, and interpretation of data or writing of the manuscript. F.B. reports personal fees from member of the external advisory board for Merck Serono, grants from Research support grant Merck Serono, outside the submitted work. A.E.P.C. reports and Unrestricted grant of Ferring B.V. to the Center for Reproductive medicine, no personal fee. Author up-to-date on Hyperthecosis. Congress meetings 2019 with Ferring B.V. and Theramex B.V. M.G. reports Department research and educational grants from Guerbet, Merck and Ferring (location VUMC) outside the submitted work. E.R.G. reports personal fees from Titus Health Care, outside the submitted work. C.B.L. reports grants from Ferring, grants from Merck, from Guerbet, outside the submitted work. The other authors have none to declare.TRIAL REGISTRATION NUMBERDutch Trial Register (Trial NL6414 (NTR6590), https://www.trialregister.nl/).TRIAL REGISTRATION DATE23 July 2017DATE OF FIRST PATIENT’S ENROLMENT10 April 2018

P–593 Self-monitoring of hormones via a urine-based hormonal assay — a topical endeavour into telemedicine in medically-assisted reproduction (MAR)

Study questionHow can cycle monitoring using a urine-based hormonal assay device improve current clinical practice in medically assisted reproduction (MAR)?Summary answerA urine-based hormonal assay has the potential to overcome the inconvenience of blood tests and reduce the frequency of appointments, waiting times and patient burden.What is known alreadyCycle monitoring via ultrasound and serum-based hormonal assays during MAR can provide information on the ovarian response and assist in optimising treatment strategies and reducing complications, such as ovarian hyperstimulation syndrome (OHSS). However, blood tests may cause inconvenience to patients due to repeated venepuncture and the need for frequent clinic appointments. Urine-based assays have been historically used by fertility specialists in clinics, but since got replaced by more practical and automated serum-based assays. Novel technology utilising rapid chromatographic immunoassay to test urinary reproductive hormones in a home setting could provide an alternative to current serum-based testing at clinics.Study design, size, durationA questionnaire was disseminated among 24 fertility specialists (2019–2020) on the use of ultrasound and serum-based hormone monitoring in clinical practice. In addition, the literature on the reliability of urine-based hormonal assays compared to serum-based hormonal assays during MAR was reviewed in order to examine if urine-based hormonal monitoring could be re-introduced in clinical practice using novel state-of-the-art technology.Participants/materials, setting, methodsAll 24 surveyed fertility specialists responded, representing 10 countries from across Europe, Asia and Latin America. Questions assessed the frequency and role of hormonal monitoring, the hormones tested and the drawbacks of blood tests. The PubMed search engine was used to search the Medline database for publications between 1960–2020 with (MeSH-) search terms related to cycle monitoring (e.g. fertility monitoring, controlled ovarian stimulation, ovulation confirmation) and hormonal assays (e.g. estrone–3-glucuronide or E1–3G).Main results and the role of chanceThe survey confirmed that many fertility practitioners (n = 22/24) routinely conducted hormone monitoring during MAR, primarily for guiding dose adjustments (n = 20/24) and indicating risk of OHSS (n = 20/24). The reported drawbacks of blood tests included validity of results from different service providers, long waiting times and discomfort to patients due to travelling to clinics for tests and repeated venepunctures. The hormones routinely checked were E2 (n = 22/22), P4 (n = 18/22) and LH (n = 15/22). The literature review revealed a relatively high correlation (correlation coefficients 0.85–0.95) between serum E2 and urinary E1–3G in gonadotrophin stimulated cycles (Lessing 1987, Catalan 1989, Rapi 1992 and Alper 1994). No studies assessed the correlation between serum P4 and urinary PdG or between serum LH and urinary LH in stimulated cycles. In natural cycles, the correlation coefficients between serum P4 and urinary PdG seemed to be slightly higher than those between serum E2 and urinary E1–3G (0.73–0.94 vs. 0.54–0.88) (Denari 1981, Munro 1991, Roos 2015, Stanczyk 1980). One study reported a moderate correlation coefficient (0.72) between serum and urinary LH in natural cycles (Roos 2015).Limitations, reasons for cautionThere is risk of selection-bias for fertility specialists included in survey, however, the 100% response rate is reassuring. The correlation coefficients between serum- and urine-based hormonal assay and the cost-effectiveness and time-efficiency of urinary assay should be confirmed in further clinical studies using a novel state-of-the-art remote urinary monitoring device.Wider implications of the findings: Remote hormonal monitoring can be part of a novel digital health solution that includes remote ultrasound and tele-counselling to link clinics and patients at home. Especially during the unprecedented times of the COVID–19 pandemic, the prospect of remote monitoring system has the potential to improve patient experience during fertility treatment.Trial registration numberNot applicable

Elevated Testosterone Levels With Unclear Etiology- the Need for Longer Follow up and Role of Dexamethasone Suppression Test as a Useful Tool to Distinguish Tumorous vs Non Tumorous Etiology

Background: Severe hirsutism in women in conjunction with elevated testosterone level raises concern for androgen secreting tumors. When initial investigations and radiological imaging do not identify a tumorous pathology, clinicians are faced with a dilemma on whether to investigate further or to consider a benign cause such as PCOS or ovarian hyperthecosis. There is inadequate evidence on how long these patients need to be followed up before considering a benign cause for their symptoms. Clinical Case: 57 years-old female, with pertinent history including primary hypothyroidism and eczema, was referred to endocrine clinic in September-2011 for work-up of severe hirsutism and elevated testosterone levels of 4.1 nmol /L (n: 0 - 2.5). All other tests including androstenedione, DHEAS, baseline pituitary profile and 24-hours urinary free cortisol levels were unremarkable. MRI of adrenal glands and ovaries was also unremarkable. Patient was presumptively diagnosed with ovarian hyperthecosis and commenced on spironolactone. There was improvement in hirsutism and patient was discharged from clinic in Feb 2012. Patient was re-referred to endocrine clinic 7 years later in September-2019 for worsening of hirsutism, male pattern baldness. At this stage, patient had testosterone levels of 17– 23 nmol/L (n: 0 - 2.5). Free androgen index 76.6% (n:0–7), SHBG (35 nmol/L, n: 18 – 114). Androstenedione (4 nmol, n: 1 – 8.5) DHEAS (2.3 umol/L, n: 0.3 – 12), 24-hours urine free cortisol level (< 13 nmol, n: < 165 nmol), 17-hydroxyprogesterone, serum ACTH, TSH, LH and FSH and estradiol levels were all normal. On examination patient had signs of virilization which had developed over previous six months. Patient had a low dose dexamethasone suppression test (0.5 mg of dexamethasone 6 hourly for 48 hours). The androgen profile obtained pre and post test showed no suppression in testosterone but well suppressed cortisol. Patient had repeat MRI of the adrenals and ovaries which revealed focal enhancing mass in right ovary (3.3 x 2.5 x 2.6 cm). Patient had an urgent bilateral oophorectomy and histology confirmed a rare steroid cell tumour of the right ovary. Following surgery there has been a significant improvement in her symptoms. Conclusion: Patients with elevated testosterone level and unclear etiology need longer follow up and review of investigations when symptoms worsen as yet undiscovered sinister etiology could be the likely reason. Dexamethasone suppression can be considered as a useful tool to distinguish tumorous vs non tumorous etiology in early stage of investigations as poor suppression of androgens with dexamethasone increases the likelihood of tumorous etiology1

Urinary gonadotropin measurements by enhanced luminometric assays (LIA) for the evaluation of pubertal development.

Determination of LH in urine has proved to be a reliable method for evaluation of pubertal development. The human LH assay based on time-resolved immunofluorometric (IFMA) technology (AutoDELFIA, PerkinElmer, Wallac) has been found to be suitable for this purpose thanks to its high sensitivity but other assays have not been evaluated. We have analyzed our data obtained by another potentially sensitive detection technique, enhanced luminometric assay (LIA) with the objective of finding a viable alternative to IFMA since these may not be available in the future. LIA was used to measure LH and FSH in serum and urine samples from 100 healthy subjects of each Tanner stage and both genders, whose pubertal development has been determined. Urinary gonodotropin concentrations measured by LIA correlated well with Tanner stage [(r=0.93 for girls, r=0.81 for boys; p<0.01 for LH) and (r=0.81 for girls, r=0.73 for boys; p<0.01 for FSH)]. LIA determinations revealed the increase in U-LH concentrations during the transition from Tanner stage 1-2 in both girls and boys (p<0.001), whereas U-FSH and S-LH were able to detect the increase from Tanner stage 1-2 only in boys or girls, respectively (both p<0.001). Measurement of urinary gonadotropin concentrations by LIA may be useful for the evaluation of overall pubertal development and also in the detection of transition from prepuberty to puberty.

The Role of Urinary LH and FSH in the Diagnosis of Pubertal Disorders.

Background:Various hormonal parameters used to differentiate between different causes of pubertal disorders are invasive, cumbersome, and has variable sensitivity and specificity. Thus, the use of a noninvasive test like urinary gonadotropin for the diagnosis of pubertal disorders will offer a significant advantage.Objective:To study the role of urinary gonadotropins (uLH, uFSH) for the diagnosis of various pubertal disorders and in the monitoring of Gonadotrophin releasing hormone, Hypothalamic-pituitary-gonadal (GnRHa) therapy in patients with central precocious puberty (CPP).Materials and Methods:We evaluated 35 healthy children and 96 patients with disorders of puberty out of which 31 cases had early puberty and 65 cases had delayed puberty. We used Spearman's correlation coefficient to evaluate the correlation between the serum and urinary gonadotropins. We used Mann–Whitney U test (for 2 groups) and Kruskal–Wallis test (for > 2 groups) to compare the median urinary and serum gonadotropins of different groups.Results:The urinary gonadotropins correlated strongly with serum gonadotropins in both healthy controls and individuals with pubertal disorders. The uLH level of ≥0.76 IU/L had 100% sensitivity and specificity to differentiate CPP from peripheral precocious puberty, whereas uLH level of ≥1.07 IU/L had 100% sensitivity and specificity for differentiating CPP from PT. In patients with delayed puberty, uFSH of ≥20.51 IU/L had 94.7% sensitivity and 91.3% specificity for the diagnosis of Hyper-Hypo cases and uLH level of ≥0.5 IU/L had sensitivity of 96.2% and specificity of 85% to differentiate constitutional delay in growth and puberty from hypogonadotropic-hypogonadism. In CPP patients on GnRHa therapy, the uLH level of ≥0.13 IU/L had 100% sensitivity and 86.7% specificity to identify those who had nonsuppressed serum LH levels.Conclusion:The urinary gonadotropins can be used as a reliable noninvasive test for the diagnosis of various pubertal disorders and also for monitoring of CPP patients on GnRHa therapy.

Fecundability in relation to use of mobile computing apps to track the menstrual cycle.

To what extent does the use of mobile computing apps to track the menstrual cycle and the fertile window influence fecundability among women trying to conceive? After adjusting for potential confounders, use of any of several different apps was associated with increased fecundability ranging from 12% to 20% per cycle of attempt. Many women are using mobile computing apps to track their menstrual cycle and the fertile window, including while trying to conceive. The Pregnancy Study Online (PRESTO) is a North American prospective internet-based cohort of women who are aged 21-45 years, trying to conceive and not using contraception or fertility treatment at baseline. We restricted the analysis to 8363 women trying to conceive for no more than 6 months at baseline; the women were recruited from June 2013 through May 2019. Women completed questionnaires at baseline and every 2 months for up to 1 year. The main outcome was fecundability, i.e. the per-cycle probability of conception, which we assessed using self-reported data on time to pregnancy (confirmed by positive home pregnancy test) in menstrual cycles. On the baseline and follow-up questionnaires, women reported whether they used mobile computing apps to track their menstrual cycles ('cycle apps') and, if so, which one(s). We estimated fecundability ratios (FRs) for the use of cycle apps, adjusted for female age, race/ethnicity, prior pregnancy, BMI, income, current smoking, education, partner education, caffeine intake, use of hormonal contraceptives as the last method of contraception, hours of sleep per night, cycle regularity, use of prenatal supplements, marital status, intercourse frequency and history of subfertility. We also examined the impact of concurrent use of fertility indicators: basal body temperature, cervical fluid, cervix position and/or urine LH. Among 8363 women, 6077 (72.7%) were using one or more cycle apps at baseline. A total of 122 separate apps were reported by women. We designated five of these apps before analysis as more likely to be effective (Clue, Fertility Friend, Glow, Kindara, Ovia; hereafter referred to as 'selected apps'). The use of any app at baseline was associated with 20% increased fecundability, with little difference between selected apps versus other apps (selected apps FR (95% CI): 1.20 (1.13, 1.28); all other apps 1.21 (1.13, 1.30)). In time-varying analyses, cycle app use was associated with 12-15% increased fecundability (selected apps FR (95% CI): 1.12 (1.04, 1.21); all other apps 1.15 (1.07, 1.24)). When apps were used at baseline with one or more fertility indicators, there was higher fecundability than without fertility indicators (selected apps with indicators FR (95% CI): 1.23 (1.14, 1.34) versus without indicators 1.17 (1.05, 1.30); other apps with indicators 1.30 (1.19, 1.43) versus without indicators 1.16 (1.06, 1.27)). In time-varying analyses, results were similar when stratified by time trying at study entry (<3 vs. 3-6 cycles) or cycle regularity. For use of the selected apps, we observed higher fecundability among women with a history of subfertility: FR 1.33 (1.05-1.67). Neither regularity nor intensity of app use was ascertained. The prospective time-varying assessment of app use was based on questionnaires completed every 2 months, which would not capture more frequent changes. Intercourse frequency was also reported retrospectively and we do not have data on timing of intercourse relative to the fertile window. Although we controlled for a wide range of covariates, we cannot exclude the possibility of residual confounding (e.g. choosing to use an app in this observational study may be a marker for unmeasured health habits promoting fecundability). Half of the women in the study received a free premium subscription for one of the apps (Fertility Friend), which may have increased the overall prevalence of app use in the time-varying analyses, but would not affect app use at baseline. Most women in the study were college educated, which may limit application of results to other populations. Use of a cycle app, especially in combination with observation of one or more fertility indicators (basal body temperature, cervical fluid, cervix position and/or urine LH), may increase fecundability (per-cycle pregnancy probability) by about 12-20% for couples trying to conceive. We did not find consistent evidence of improved fecundability resulting from use of one specific app over another. This research was supported by grants, R21HD072326 and R01HD086742, from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, USA. In the last 3 years, Dr L.A.W. has served as a fibroid consultant for AbbVie.com. Dr L.A.W. has also received in-kind donations from Sandstone Diagnostics, Swiss Precision Diagnostics, FertilityFriend.com and Kindara.com for primary data collection and participant incentives in the PRESTO cohort. Dr J.B.S. reports personal fees from Swiss Precision Diagnostics, outside the submitted work. The remaining authors have nothing to declare. N/A.

Self-Detection of the LH Surge in Urine After GnRH Agonist Trigger in IVF-How to Minimize Failure to Retrieve Oocytes.

Research question: Urine LH testing may be useful to confirm an LH surge after the GnRH agonist (GnRHa) trigger prior to oocyte retrieval in IVF.Design: A prospective cohort study, including oocyte donors undergoing ovarian stimulation, treated with a GnRHa trigger for final oocyte maturation. Urine LH testing was performed at home, 12 h after the GnRHa trigger. In the case of a negative result, serum LH and progesterone measurements were done that same day. Donors with no serum LH peak after trigger were re-scheduled using a dual trigger, with GnRHa and hCG.Results: Three hundred and fifty nine oocyte donors were included in the analysis. Three hundred and fifty six donors had positive urine LH tests, followed by oocyte retrieval. In one case, the LH test was positive, however, no oocytes were retrieved (false positive 1/356). Three LH tests were negative in urine: in one of these three cases, LH was tested again in blood, confirming an LH rise, consistent with an optimal response to the GnRHa trigger; in the other two cases, serum LH was <15 mUI/mL, after which the oocyte retrieval was re-scheduled for 36 h after an being re-triggered, resulting in the retrieval of 19 and 22 MII oocytes, respectively. Considering the cost analysis, it would be a significantly cost-saving strategy, as blood testing would have costed 14,840€ vs. only 185.5€ in urine LH kits.Conclusions: Urinary testing of the LH surge after GnRHa trigger is easy, safe, reliable, and convenient. In addition, LH urine testing allows identifying donors and patients who could benefit from a rescue hCG trigger after an unsuccessful GnRHa trigger.