Urinary hCG Research Articles

A-337 The Utility of Urinary Human Chorionic Gonadotropin Testing in the Emergency Department

Abstract Background Human chorionic gonadotropin (hCG) levels are measured in both urine and blood as an indication of suspected pregnancy, and assessment of progress of conception. It is also measured in the investigation of patients presenting with acute abdomen and suspected of ectopic or molar pregnancy. This study reviewed the utility of urine pregnancy test in a large university teaching hospital. Methods Results of all urinary hCG tests (using hCG Combo Pregnancy kit, MEDLINE, IL) performed as point of care in the emergency department during the year 2023 were obtained. Corresponding plasma hcG levels performed (Total β-HCG Alinity immunoassay, Abbott Diagnostics, IL) were also obtained. Analysis of hCG testing outcomes were assessed. Results A total of 2848 urinary hCG tests were performed during the study period. The majority 2113 (74.2%) of samples were negative for hCG whereas 835 samples (29.3) were positive. Among the positive hCG urine samples, 401 patients (48%) had plasma hCG requested and performed. Plasma hCG values ranged from < 2.4 (26.5) to 219,462.5 mIU/mL (median 16,872.35 mIU/mL). One plasma sample with corresponding positive urinary hCG result had plasma hCG level <2.4 mIU/mL which is below the detection limit of the assay suggesting a false positive urinary hCG result. Among the 13 patients with serial plasma hCG values, only one patient exhibited doubling (48 hours) hCG values when less than 13,000 mIU/mL. One patient had a declining serial hCG plasma values suggesting failing conception. The remaining patients with consecutive plasma hCG values cannot be assessed as either the time gap was excessive or hCG values were markedly elevated (> 30,000 mIU/mL). Conclusions Although the utility of urinary hCG testing in the emergency department appeared appropriate, unnecessary repeat analysis with appropriate plasma request represented 3.2%. An electronic test request duplicate notification may help resolve this unnecessary repeat.

Evaluation of Jacalin lectin sorbents for the extraction of the human chorionic gonadotropin glycoforms prior to analysis by nano liquid chromatography-high resolution mass spectrometry

Human chorionic gonadotropin (hCG) is a dimeric, highly glycosylated hormone with a total of 4 N- and 4 O-glycosylation sites in its two subunits, hCGα and hCGβ. Recently, we developed a novel nano liquid chromatography coupled to high resolution mass spectrometry (nanoLC-HRMS) method for the analysis and thus the detection of the intact glycoforms of hCG. Here, a sorbent functionalized with the Jacalin lectin was evaluated in solid-phase extraction (SPE) for its potential to fractionate the hCG glycoforms prior to their nanoLC-HRMS analysis at the intact level, which may facilitate the detection of low-abundance glycoforms and may lead to a more detailed characterization of the hormone glycosylation. A commercial sorbent based on Jacalin immobilized on Sepharose and having a lectin density of 4.5mg per ml of gel was selected to carry out SPE and its capacity was estimated to be of some tens of μg of hCG per ml of lectin sorbent. Next, the SPE protocol was modified to improve the extraction recoveries. Especially, it was noticed that an extensive pre-conditioning procedure prior to the first use of a cartridge was necessary to remove the residual non-grafted lectins. Indeed, if on-grafted lectins are not eliminated, they may bind a part of hCG glycoformes preventing they retention by the sorbent, leading to low extraction recoveries (around 10%). With the extensive pre-conditioning procedure, the average extraction recoveries for both hCGα and hCGβ glycoforms were about 50%, with either recombinant or urinary hCG. Qualitatively, the fractionation of hCG glycoforms between the washing and elution fractions was achieved with the urinary hCG sample by determining the number of glycoforms detected in each fraction. It appears that 12 hCGα glycoforms have a low affinity (detected only in the washing fraction), 1 a low-medium affinity (detected in washing and elution 1 fractions), 16 a medium affinity (detected in washing, elution 1 and 2 fractions), and 12 a high affinity (detected only in elution 1 and 2 fractions). For the hCGβ glycoforms, similarly, 3 have a low affinity and 12 a low-medium affinity. Additionally, the 3 hCGβ glycoforms were detected better. A different behavior was observed with the recombinant hCG sample, which indicates glycosylation differences between the two hCG samples. This shows the potential of lectin-based affinity fractionation before nanoLC-HRMS analysis to better characterize the glycosylation state of hCG at the intact level.

Analytical Investigation of the Profile of Human Chorionic Gonadotropin in Highly Purified Human Menopausal Gonadotrophin Preparations.

Highly purified human menopausal gonadotropin (HP-hMG [Menopur®, Ferring Pharmaceuticals, Saint-Prex, Switzerland]) contains a 1:1 ratio of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). This analysis aimed to assess gonadotropin (FSH, LH and hCG) abundance in HP-hMG and clarify the source of hCG by assessing the presence of sulfated glycans, which are diagnostic for pituitary hCG forms due to their distinct glycosylation patterns. Additionally, the purity of each sample, their specific components, and their oxidation levels were assessed. HP-hMG samples (three of Menopur® and two of Menogon® Ferring Pharmaceuticals, Saint-Prex, Switzerland) were included in the current analyses. Brevactid® (urinary hCG; Ferring Pharmaceuticals, Saint-Prex, Switzerland) and Ovidrel® (recombinant hCG; Merck KGaA, Darmstadt, Germany) were used as control samples. Glycopeptide mapping and analysis of impurities were carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Oxidation was assessed through reducing peptide mapping using LC-MS/MS. The FSH and LH in the HP-hMG samples showed sulfated glycans, while no signals of sulfated glycopeptides were detected on any site of the beta subunit of hCG. HP-hMG test samples presented the same hCG glycan distribution as the control sample (placental hCG, Brevactid®) extracted from the urine of pregnant women, suggesting a non-pituitary source of hCG. Protein impurities were estimated to constitute approximately 20-30% of the entire HP-hMG protein content in the test samples. More than 200 non-gonadotropin proteins were identified in the HP-hMG test samples, of which several were involved in embryonic development or pregnancy. The alpha subunit of the tested samples was strongly oxidized, with a relative abundance of 20% of the total gonadotropin content. Without taking into account all the protein impurities, the beta subunit of LH was detected only in traces (0.9-1.2%) in all tested HP-HMG samples, confirming the data obtained by intact molecule analysis, while high levels of beta hCG (18-47%) were observed. Advanced molecular analysis of HP-hMG indicates a primarily placental origin of hCG, as evidenced by the absence of hCG sulfated glycans and the predominance of placental non-sulfated hCG in LH activity. The analysis revealed 20-30% of protein impurities and a significant presence of oxidized forms in the HP-hMG samples. These findings are critical for understanding the quality, safety, and clinical profile of HP-hMG.

P-703 Is the time interval between triggering final oocyte maturation and oocyte retrieval associated with ART outcomes? A systematic review and meta-analysis

Abstract Study question Is the time interval between triggering final oocyte maturation and oocyte retrieval associated with assisted reproductive technology (ART) outcomes? Summary answer Limited evidence suggests that extending the time interval between triggering final oocyte maturation and oocyte retrieval from 33-36h to 37-41h does not affect ART outcomes. What is known already The time interval between final oocyte maturation and oocyte retrieval varies among IVF centres, although an interval of 33–36 h is the most widely adopted. The use of a prolonged time interval of 37-41h has been suggested to increase the number and competence of oocytes retrieved, their maturation, their ability to be fertilized, their capacity to produce usable embryos and the probability of pregnancy. Considering recently published data, a systematic review is warranted to provide clinicians with updated, evidence-based information regarding the optimal time interval between triggering final oocyte maturation and oocyte retrieval. Study design, size, duration A systematic review and meta-analysis was performed aiming to evaluate the effect of the time interval between triggering final oocyte maturation and oocyte retrieval on ART outcomes. For this purpose, a literature search was carried out until January 2024 in PubMed, MEDLINE and CENTRAL. The primary outcome measure was clinical pregnancy rate, while ovulation before oocyte retrieval, oocytes retrieved, maturation rate, fertilization rate, blastulation rate, implantation rate and miscarriage rate were secondary outcomes. Participants/materials, setting, methods Citation, demographic, methodological and clinical data were extracted from the studies by two independent reviewers. Quality was assessed using the RoB2 Tool by Cochrane. Statistical heterogeneity was assessed by I2. In dichotomous data, estimates were expressed as relative risk (RR) with 95% confidence intervals (CIs), using the fixed or random effects method. In continuous data, differences were pooled across resulting in a weighted mean difference (WMD) with 95% CI. Main results and the role of chance Six eligible RCTs (n = 1081) were identified, published between 1991-2023. In three trials, conventional insemination was used for fertilization, in two trials this was performed by intracytoplasmic sperm injection (ICSI), whereas in one trial either conventional insemination or ICSI were used. The time intervals between triggering final oocyte maturation and oocyte retrieval compared, varied between studies (34.5-36 vs 38.5-40h, 33 vs 41h, 34 vs 38h, 35 vs 37h, 34 vs 39h and 36 vs 38h). Patients were grouped in advance based on these time intervals (short interval group: 33-36h versus long interval group: 37-41h). Ovarian stimulation was performed with GnRH analogues (GnRH antagonists:1 GnRH agonists:5) and gonadotrophins (HMG:4 recombinant FSH:2, while triggering of final oocyte maturation was performed with hCG( recombinant hCG:1 urinary hCG:5). None of the studies was deemed as of low risk of bias. No statistically significant differences were observed between the short nd long interval groups regarding clinical pregnancy rate per randomized patient (RR: 0.83; 95%CI: 0.62-1.11; fixed effects model; heterogeneity: I2:36.4%; five studies, 827 patients). Furthermore, it was not feasible to pool any of the secondary outcome data. This was either because they were not reported, or due to the fact that they were reported inappropriately. Limitations, reasons for caution This meta-analysis included a limited number of RCTs, which were generally of low quality due to methodological limitations and of high risk of bias. Moreover, the total number of patients is still not large enough to draw solid conclusions. Wider implications of the findings Taking into account the limitations of the current meta-analysis, it appears that clinicians may elect a shorter (33–36 h) or a longer (37-41h) interval regarding the timing of oocyte retrieval following triggering final oocyte maturation, without affecting clinical pregnancy rate. Trial registration number PROSPERO: CRD42024501986

Human chorionic gonadotropin regulates cytokine production by lymphocytes from patients with multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) that primarily affects young adults, predominantly females. This was partially attributed to sex differences in immunity, which are influenced by changes in sex hormones occurring during women‘s life, among other factors. Furthermore, MS patients experience significant improvement in their symptoms during pregnancy when levels of female sex-hormones significantly increase. This phenomenon was attributed to immune adaptations occurring during gestation which are regulated by paternal antigens and sex hormones. The human chorionic gonadotropin (hCG) was shown to have strong immunosuppressive abilities. We aimed to analyze here the capacity of the hCG to regulate pro- and anti-inflammatory cytokine production by PBMC from MS patients. PBMC isolated from 17 MS patients receiving IFNβ1a treatment were cultured with or without recombinant or urinary hCG. Cytokine production in the supernatants was assessed using a CBA array and cytokine production by lymphocytes and expression of co-stimulatory molecules in B-lymphocytes were evaluated by flow cytometry. hCG reduced the production of TNF by PBMC from MS patients while lowering the percentages of TNF producing T cells and diminishing the production of TNF by B cells. hCG significantly boosted the production of IL-10 by regulatory T cells and CD19high B cells from MS patients. Furthermore, hCG treatment lowered the percentages of CD80+CD86+ expressing B cells within PBMC from MS patients. Overall, our results described a novel and not yet explored mechanisms of action of hCG in the context of MS.

Role of Granulocyte Colony Stimulating Factor Effects on Unresponsive Thin Endometrium in Women Undergoing Controlled Ovarian Stimulated Intrauterine Insemination Cycles

Back Ground: It is a prospective observational study that included unexplained infertility cycles with COS –IUI protocols. Thin endometrium of less than 7mm has a negative effect on pregnancy rate. Endometrial function and receptivity factors are important in the success of implantation.
 Aim: To monitor the effects of G-CSF on thin endometrium in improving endometrium thickness and pregnancy rates in G-CSF administered COS-IUI cycles.
 Methods: This study was conducted in the Laxmi Narasimha hospital IVF center under the guidence Dr. Adaboina Anitha,Hanamkonda, warangal. Thin endometrium was defined less than 7mm on Trans vaginal ultrasound. Letrozole 5mg (2.5mg, BD) is used for ovulation induction on day 2 or day 3 of their cycle, G CSF 300units was added with significantly improved pregnancy rates in ART cycles. Trigger used was injection 10,000IU urinary hCG & 300 units G-CSF was instilled into the uterus. After 48hours ET was measured & IUI was performed under aseptic precautions. After 16 days β -hCG levels were monitored to determine whether there is a pregnancy.
 Results: In present study, we concluded that, 250 COS-IUI cycles were analysed and 50 cycles showed a thin endometrium and in them G-CSF was used. The pregnancy rate (28%) was evaluated, 14 members are positive for pregnancy.
 Conclusion: In our study we concluded that G-CSF and letrozole will improve increases endometrium thickness and conceiving rates in COS-IUI cycles.

A randomized, controlled clinical study of low-molecular-weight heparin improving pregnancy outcomes in PCOS women undergoing IVF: study protocol

BackgroundPolycystic ovary syndrome (PCOS), an incidence of 10–15% in women of reproductive age, shows sex hormone disorders, luteal insufficiency, and the tendency of placental villus space thrombus. The incidence of early pregnancy loss in women with PCOS is three to eight times higher than that in non-PCOS women. PCOS women were reported in a pre-thrombotic state, which was manifested by accelerated thrombin production, increased PAI-1 activity, and fibrinogen. Other research also found an over-activated state of women with PCOS in immune system. Therefore, changing the prethrombotic state of PCOS through anticoagulation may be a new way to improve the adverse pregnancy outcome of PCOS. Low-molecular-weight heparin (LMWH) is the most common used anticoagulant drug in pregnancy, and it also was proposed for the prevention of recurrent abortion, although the application of LMWH in PCOS population during early pregnancy has not been reported. The objective of this study is to investigate the effect of LMWH on pregnancy outcomes after invitro fertilization-frozen embryo transfer (IVF-FET) in patients with polycystic ovary syndrome.MethodsA total of 356 PCOS women aged between 20 and 38 years which prepared for IVF followed with FET will be enrolled in the study. The patients, from four different hospitals stratified by age and body mass index (BMI), will be randomly divided into the study group who will be treated with LMWH started on the day of progesterone transformation (hormone therapy) during FET cycle and the control group without additional medicine. Serum or urine hCG test will be given 14 days after embryo transfer to confirm biochemical pregnancy. If pregnancy is positive, LMWH+ hormone therapy/hormone therapy will be continued for another 2 weeks. Transvaginal ultrasonography will be performed 14 days later to confirm intrauterine pregnancy. The primary outcome is the ongoing pregnancy, which is defined as intrauterine live fetus with ultrasound after 12 weeks of gestation.DiscussionThis is the first study protocol to investigate the efficacy of LMWH as an adjuvant drug for IVF-FET outcomes in PCOS women, by comparing differences in ongoing pregnancy rate, clinical pregnancy rate, live birth rate, and early pregnancy loss rate between LMWH group and the control group.Trial registration ClinicalTrials.gov ChiCTR2000036527. Registered on August 24, 2020

A-350 Myth Debunked: Same UrineAliquot is not Suitable for hCG Test AfterMicroscopic Analysis

Abstract Background It has been imparted in the laboratories that the suspending fluid, Lamina, from Iris IQ 200 Analyzer system is prone to interfere with urine human chorionic gonadotropin (hCG) test, especially on a small volume urine specimen. The myth is that the sample is diluted or contaminated by Lamina, which gives falsely negative result for hCG test. An add-on urine pregnancy test was ordered for a specimen, which was completed urinalysis and microscopic analysis. This is the only urine aliquot available, and it is approximately 2mL. This order raised many concerns among laboratory technicians regarding the accuracy of the result. However, no documents or articles were found to support the suspicions. In this study, A serial of experiments was completed to address this concern. Methods Known pregnant patients were confirmed their pregnancies with serum hCG tests prior to collecting the urine specimens. The experiment was conducted with 4 positive patient urines. A series of samples ran on DXI 800 system and the point-of-care (POC) Alere hCG combo cassettes. Then the same specimen performed urinalysis and microscopic analysis on Iris IQ 200 Analyzer. To evaluate the Lamina impact, the sample concentrations were verified with point-of-care (POC) Alere hCG combo cassettes and later DXI 800 system. Meantime, the significance of sample size was also evaluated in the study. Results The final concentrations of the samples were at <5, 5, 10, 15, 20, 25, 30, 40, 50, and 60 mIU/mL. The cutoff for Alere hCG combo cassettes was verified at 20 mIU/mL for urine, the assay gives the accurate result at and above that value. The results are consistent between immunoassay and POCT prior to and after microscopic analysis. Therefore, no significant interferences were identified with the desired sample volume, 3 mL, or short sample volume, 2mL. The volume usage by iQ 200 Analyzer was less than 500 uL. Conclusion In this study, Lamina doesn't cause any interference with hCG values using Alere hCG combo cassettes despite the short sample volume. However, variations between POC urinary hCG devices may contribute to different outcomes. Therefore, a separate validation is highly recommended if a different POC urinary hCG device is involved.

Beyond the Umbrella: A Systematic Review of the Interventions for the Prevention of and Reduction in the Incidence and Severity of Ovarian Hyperstimulation Syndrome in Patients Who Undergo In Vitro Fertilization Treatments.

Ovarian hyperstimulation syndrome (OHSS) is the main severe complication of ovarian stimulation for in vitro fertilization (IVF) cycles. The aim of the current study was to identify the interventions for the prevention of and reduction in the incidence and severity of OHSS in patients who undergo IVF not included in systematic reviews with meta-analyses of randomized controlled trials (RCTs) and assess and grade their efficacy and evidence base. The best available evidence for each specific intervention was identified, analyzed in terms of safety/efficacy ratio and risk of bias, and graded using the Oxford Centre for Evidence-Based Medicine (CEBM) hierarchy of evidence. A total of 15 interventions to prevent OHSS were included in the final analysis. In the IVF population not at a high risk for OHSS, follitropin delta for ovarian stimulation may reduce the incidence of early OHSS and/or preventive interventions for early OHSS. In high-risk patients, inositol pretreatment, ovulation triggering with low doses of urinary hCG, and the luteal phase administration of a GnRH antagonist may reduce OHSS risk. In conclusion, even if not supported by systematic reviews with homogeneity of the RCTs, several treatments/strategies to reduce the incidence and severity of OHSS have been shown to be promising.

From Cereal Grains to Immunochemistry-What Role Have Antibodies Played in the History of the Home Pregnancy Test.

Today, the home pregnancy test is the most frequently performed laboratory test for self-diagnosis (home diagnostic test). It is also the first laboratory test that has been adapted for self-use at home. This is probably because women have always wanted to know the answer to the question: "Am I pregnant or not?" and always preferred to know the answer to this question intimately and in a discreet way. The history of the pregnancy test is also an interesting example of how the discovery of antibodies and the development of in vitro diagnostic methods based on the antigen-antibody reaction were important for the development of laboratory and clinical diagnostics. Immunodiagnostic techniques (based on the antigen-antibody reaction) are currently the basis of modern specialist laboratory diagnostics, which is essential in clinical diagnosis. The history of the pregnancy test is an interesting one and dates back to ancient times. A pregnancy test is defined as a procedure intended to reveal the presence or absence of pregnancy. Nowadays, every pregnancy test is based on the detection of human chorionic gonadotropin (hCG) in urine or blood. Human chorionic gonadotropin is secreted by the placenta right after a fertilized egg cell implants in the uterus and can be detected in both the urine and blood of pregnant women. Urine pregnancy tests are convenient for self-use at home. Blood tests are performed in medical laboratories. Specialized laboratory methods not only detect hCG but also determine the concentration of this hormone. However, both of these methods are highly accurate and common. Throughout the ages, many different methods were used to detect pregnancy at the earliest stage. Grain, wine, and various small animals were used as research tools. These were both long-term and often unreliable; most were based on folk beliefs and superstitions. Animal pregnancy tests were the first biological tests used in this field. This was a significant advance in the accurate detection of relatively early pregnancy. Animal tests in modern times are considered cruel and inhumane, no matter how reliable their results can be. Their place is now taken by much more specific, more sensitive, and definitely more ethical immunochemical tests. The pregnancy test and the methods to find out whether a woman is pregnant have gone through massive transformations, from bioassays using plants to bioassays on animals to advanced immunochemical techniques and biosensors. Modern pregnancy tests are not invasive and are very sensitive. Nowadays, it takes only about 3 min to know the answer to the question: "Am I pregnant or not?". However, it was not always as simple as it is today. This manuscript aims to show the important role played by antibodies in the development of laboratory and clinical diagnostics in the example of the interesting history of the pregnancy test.

A semi-automated hybrid HPLC-MS approach for in-depth characterization of intact non-covalent heterodimer glycoforms of gonadotropin biopharmaceuticals

BackgroundGonadotropins are a class of heavily glycosylated protein hormones, thus extremely challenging to characterize by mass spectrometry. As biopharmaceuticals, gonadotropins are prescribed for the treatment of infertility and are derived from different sources: either from pooled urine of pregnant women or upon production in genetically modified Chinese Hamster Ovary cells. Human chorionic gonadotropin (hCG) is sold as a biopharmaceutical under the name Pregnyl® (urinary hCG, u-hCG) and Ovitrelle® (recombinant hCG, r-hCG), and recombinant human follicle stimulating hormone (r-hFSH) is marketed as Gonal-f®. Recently, we reported the exhaustive characterization of r-hCG at different structural levels. ResultsWe implement size exclusion (SE) HPLC-MS to automatize the acquisition of native mass spectra of r-hCG dimer, but also u-hCG and r-hFSH, comparing the drug products up to intact heterodimer level. A hybrid HPLC-MS approach was employed for the characterization of r-hCG, u-hCG and r-hFSH drug products at different structural levels. Released glycans were analyzed by porous graphitized carbon (PGC)-HPLC-MS/MS, glycopeptides by reversed-phase (RP)-HPLC-MS/MS, subunits by RP-HPLC-MS and finally the intact native heterodimers by semi-automated online buffer exchange SE-HPLC-MS. The data were integrated using bioinformatic tools, to finally unravel the composition of 1481 co-existing dimeric glycoforms for r-hCG, 1167 glycoforms for u-hCG, and 1440 glycoforms for r-hFSH, and to compare critical quality attributes of the different drug products such as their degree of sialylation and O-glycosylation. Significance and noveltyThe strong alliance of bioanalytics and bioinformatics data integration at the different structural levels allowed the identification of more than thousand different glycoforms of r-hCG, u-hCG, and r-hFSH. The results showed that these biopharmaceuticals differ considerably in their glycosylation patterns and highlight the importance of in-depth characterization of biopharmaceuticals for quality control.© 2017 Elsevier Inc. All rights reserved.

P-374 Impact of different luteal phase support protocols in fresh embryo transfers on perinatal outcomes of singleton in vitro fertilization pregnancies: a three-decade experience.

Abstract Study question How can stimulation of corpus luteum in the luteal phase with human chorionic gonadotropin (hCG) improve obstetric and perinatal outcomes? Summary answer There is currently no evidence that the use of hCG as an alternative treatment for luteal-phase support (LPS) improves obstetric and perinatal outcomes. What is known already There are different approaches for providing LPS, although none have shown differences in pregnancy rates. However, emerging evidence has also revealed the important role of corpus luteum in obstetric and perinatal outcomes after in vitro fertilization (IVF) (preeclampsia or intrauterine growth restriction, among others) in the production of progesterone (can be administered exogenously) as well as the secretion of multiple vasoactive active substances. Study design, size, duration We performed a retrospective, single centre study between 1991-2021. A total of 2592 singleton pregnancies were analysed after fresh embryo-transfer according to LPS. Participants/materials, setting, methods IVF singleton pregnancies were stratified into two cohorts according to the LPS protocol: 1) trigger with 5000 IU urinary hCG (U-hCG) and LPS protocol with progesterone 200 mg x3/day/vaginally beginning the day after oocyte retrieval and one u-hCG bolus of 2500 IU, the day and 3 days after oocyte retrieval; and 2) trigger with 250 µgr of recombinant hCG and LPS protocol with only progesterone (200 mg x3/day/ vaginally). Main results and the role of chance We compared the reproductive outcomes between cycles in cohort 1) n = 527 and cohort 2) n = 2065. The two cohorts were stratified and analysed in three groups according to maternal age to reduce possible bias: 35 years old or less, between 36 and 39 years old, and 40 or more years old. In cohort 1, all the cycles followed a long agonist GnRH protocol, while in cohort 2, 78.11% received an agonist GnRH protocol and 21.89 % a GnRH antagonist protocol. Recombinant gonadotropins for ovarian stimulation was administered in all cycles. We observed a greater number of eutocic deliveries, a lower number of instrumentalized births and a stable caesarean rate along the study period. There were no statistical differences between the two treatment groups regarding hypertensive pregnancy disorders, intrauterine growth restriction, small for gestational age or large for gestational age. Group 2 showed an apparent greater tendency, albeit not significant, to preeclampsia and a significant increase in very preterm birth. Emerging evidence has also revealed the important role of corpus luteum in obstetric and perinatal outcomes following IVF. Limitations, reasons for caution The large number of pregnancies evaluated is a clear strength of this study. This is the first series comparing different perinatal outcomes according to LPS. In contrast, the retrospective design and long time period involving changes in laboratory and patient profiles is a clear limitation. Wider implications of the findings The optimization of IVF treatments is especially important and is related to multiple factors, including the individualization of treatments to ensure the safety of IVF treatment as well as that of the pregnancies derived thereof. More studies on the different LPS protocols available are needed. Trial registration number not applicable

O-294 Dual trigger versus hCG-only trigger in ICSI patients: an analysis of 8500 cycles

Abstract Study question Among patients undergoing ovarian stimulation for ICSI, does dual trigger with hCG and GnRH agonist offer any benefit over mono trigger with hCG alone? Summary answer Dual trigger is associated with a higher number of mature oocytes without any impact on the pregnancy rate. What is known already In ART cycles, final oocyte maturation and resumption of meiosis are generally triggered by the administration of hCG as a surrogate for the natural LH surge. Several studies have investigated the role of a combination of a bolus of GnRH agonists and hCG to mimic the natural peak of endogenous LH and FSH, without clear answers regarding clinical benefit. A meta-analysis showed highernumbers of good-quality embryos and increased ongoing pregnancy rate after dual trigger and a recent systematic review demonstrated significantly higher live birth rate (LBR) per cycle after administration of dual trigger compared to hCG-only trigger. Study design, size, duration The study was a retrospective, single-centre cohort study. In this study, 8,525 ICSI cycles were included between January 2017 and April 2022 at a tertiary referral University Hospital. We included all patients undergoing ICSI in a GnRH antagonist ovarian stimulation cycle. The mono trigger consisted of recombinant or highly purified urinary hCG. In the dual trigger group, a bolus of GnRH agonist was combined with the hCG trigger. Participants/materials, setting, methods Our cohort of 8,525 cycles was divided into two groups: Group A, the mono trigger group, (7,022 cycles), and Group B, the dual trigger group (1503 cycles). Patients who underwent IVF, pre-implantation genetic testing, oocyte donation, and fertility preservation were excluded. Patients with uterine anomalies and endocrine disorders were also excluded, as well as patients who received a triptorelin-only trigger because of hyperresponse. Main results and the role of chance There was no difference in the mean age between the dual vs mono trigger cohorts (35.93 ± 4.90 vs 35.52±4.85). The most common indication for ART was male factor infertility in both groups (21.36% in group B vs 27.11% in group A). Stimulation was shorter in the dual trigger cohort (10.41 vs 12.08 p = 0.04). The total number of cumulus oocyte complexes (8.26±5.32 vs 7.45±4.68, p < 0.001), mature oocytes (6.39±4.15 vs 5.99±3.80, p = 0.006) and fertilized oocytes (4.79±3.59 vs 4.33±3.27, p = <0.001) was higher in the dual trigger group compared to those in the mono trigger group. Day 5 embryo transfer was more prevalent in the dual trigger group (46.07% vs 33,84%, p < 0.001). Embryo utilization rate was higher in group A (61.37% ±31.98) than in group B (53.47% ±30.91 p = <0.001). Ongoing regnancy rate was similar in both groups (27.29% in group A vs 27.08% in group B). Pearson chi2, Mann Whitney and logistic regression tests were used for the analysis. Limitations, reasons for caution In spite of the large sample, this study is retrospective and holds the possibility of unmeasured confounders. Wider implications of the findings Although a dual trigger may increase the number of mature oocytes, this does not translate into a higher pregnancy nor live birth rate. Reduced oocyte competence of the “surplus” oocytes may underlie this observation. Further research should identify specific subgroups that may benefit from dual trigger across meaningful outcome parameters. Trial registration number not applicable

Impact of Biotin Supplementation on Human Chorionic Gonadotropin Immunoassays Utilizing Biotin-Streptavidin Binding Methods in Urine.

Human chorionic gonadotropin (hCG) detection is indicative of pregnancy and can be indicative of some forms of cancerous tumors. The hCG drug itself, however, is a performance enhancing substance used by male athletes to increase testosterone production. Antidoping testing for hCG is conducted in urine, often on immunoanalyzer platforms, many of which utilize biotin-streptavidin dependent immunoassays in which the presence of biotin in samples is a known confounding factor. While biotin interference in serum has been well-studied, the extent of biotin interference in urine has not. Ten active male individuals underwent a 2-week hCG administration protocol concurrent with supplementation with biotin (20 mg/day) or placebo. Urine and serum samples were collected throughout the study and analyzed for hCG and biotin concentrations. Urinary biotin levels in the hCG + biotin group increased 500-fold over baseline and 29-fold over corresponding serum biotin levels after biotin supplementation. When using a biotin-dependent immunoassay, the hCG + placebo group produced hCG-positive results (hCG ≥ 5 mIU/mL) in 71% of urine samples, while the hCG + biotin group produced positive results in only 19% of samples. Both groups had elevated hCG values in serum measurements by a biotin-dependent immunoassay and in urine when using a biotin-independent immunoassay. Urinary hCG measurements and biotin levels from the hCG + biotin group showed a negative correlation (Spearman r = -0.46, P < 0.0001) when measured using a biotin-dependent immunoassay. Biotin supplementation can severely suppress urinary hCG values in assays utilizing biotin-streptavidin binding methods and therefore these types of assays are not recommended for use in urine samples containing high levels of biotin. Clinicaltrials.gov Registration Number: NCT05450900.

Abstract 3998: Metronomic gemcitabine plus neratinib effectively inhibits the growth of human Her-2 positive breast cancer cells intracranially implanted into immunodeficient mice

Abstract Metastatic breast cancer is a major cause of death for women in the US, and breast cancers that metastasize to the brain have a particularly poor prognosis. To treat brain metastases of Her-2 positive breast cancers a number of Her-2 targeting drugs are now clinically available, including lapatinib, neratinib and tucatinib, among others. However, in part as a consequence of poor penetration of many drugs into the brain, combinations of standard chemotherapy plus targeted agent have only modest impact on progression free survival for such patients. We have developed preclinical models of human Her-2 positive breast cancer brain metastases to help address these issues. We selected human MDA-MD-361 and BT474 breast cancer cells lines by in vivo passaging in SCID mice, followed by molecular tagging of the derived variants (MDA361R and BT474R respectively), and then the stereotactic implantation of such cells into the brain of female SCID mice. Using luciferase-tagged BT474R we implanted 75,000 cells into the brains of the mice, and thereafter confirmed by luminescence the growth of the cells, which allowed for randomization of the mice to receive control saline (n=7), metronomic gemcitabine (100mg/kg/3days; i.p.; n=8), neratinib (40mg/kg/3 days; p.o.; n=7), or the combination of gemcitabine plus neratinib (n=8), which continued for 5 months largely in the absence of overt toxicity. The gemcitabine plus neratinib combination prolonged survival over the controls (150 days vs 65 days, p&amp;lt;0.5), although eventually all mice succumbed to disease. Tumors cells have been isolated from these therapy-administered mice for further characterization. Neratinib alone did not result in an increase in survival compared to controls, whereas metronomic gemcitabine improved survival but was inferior to the combination. We confirmed these findings using MDA361R tagged with human chorionic gonadotropin (hCG), which allows for the growth of cells to be monitored by assessment of hCG in the mouse urine, and these cells were implanted intracranially into female SCID mice. After urine hCG levels confirmed the presence of growing cells in the brain, the mice were randomized to the above 4 therapies (n=4/group), which showed an increase in survival of the combination compared to controls. These results point to intriguing effects of chemotherapy when used at continuous low (metronomic) doses, that in these studies involved gemcitabine administration every 3 days, and which in these models can be effectively combined with neratinib to suppress the growth of Her-2 positive breast cancer cells implanted into the mouse brain. These results highlight promising combination that could be used to suppress Her-2 positive breast cancer brain metastatic growth, and the developed models should facilitate the study of this subset of breast cancers. Citation Format: Serina Batson, Alejandro Sanchez, Hector M. Padilla, Diana L. Prospero, Brenda Lugo, Valeria V. Lopez, Daniella E. Estrada, Nydia De Avila, Saeedeh Darvishi, Arlene Levario, Shan Man, Ping Xu, Robert S. Kerbel, Guido Bocci, Giulio Francia. Metronomic gemcitabine plus neratinib effectively inhibits the growth of human Her-2 positive breast cancer cells intracranially implanted into immunodeficient mice. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3998.

Is Preoperative Urine Human Chorionic Gonadotropin (hCG) Testing Necessary for Pediatric Patients Before Oral and Maxillofacial Surgery Procedures With Sedation?

Human chorionic gonadotropin (hCG) testing is performed prior to surgical procedures to ensure patient and fetal safety. The purpose of this study was to evaluate the utility of routine pregnancy testing prior to elective outpatient oral and maxillofacial surgery procedures being performed with intravenous sedation (IVS). A retrospective cohort study was implemented assessing hCG testing in postmenarche females who underwent elective outpatient oral surgery procedures scheduled with IVS at a tertiary care institution. Medical records were used to identify eligible subjects aged 12 to 45years. The primary predictor variable was age, and the primary outcome variable was urine hCG test result. Age was divided into groups to reflect early adolescence (12 to 14years), mid-adolescence (15 to 17years), late adolescence/early adulthood (18 to 24years) and adulthood (25+ years). Secondary outcome variables included inability to void for hCG testing, change in anesthetic, case cancellation or rescheduling and were measured over a 2year period. Descriptive statistics were performed. Relative risk (RR) and Cochran-Armitage test for trend were calculated to determine the statistical significance of age on inability to void. The sample consisted of 5,006 females, with a median age (IQR, range) of 18.0 (3.6, 12.0 to 43.6) years. There was one positive urine hCG result providing a preoperative pregnancy rate of 0.02%. Fourteen of 1,195 subjects (1.2%) over a 2year period were unable to provide a urine hCG sample. There was a statistically significant trend in inability to void as age groups got older (P=.001). Patients aged 12 to 17years had an increased risk of being unable to void compared to patients 18years and older (RR: 14.30, 95% CI: 1.88 to 108.99, P=.01). The total cost of testing over the 11year observation period was $9,019.59. The risk of surgical cancellations and delayed care due to patients' inability to void preoperatively plus a lack of any positive preoperative urine hCG findings in patients under 18years of age in this study, call into question the necessity of routine preoperative hCG screening in pediatric patients presenting for IVS for elective outpatient oral and maxillofacial procedures.

LBODP011 Pembrolizumab Induced Hypophysitis And Resultant Isolated ACTH Deficiency Presenting As Secondary Adrenal Insufficiency.

Abstract Introduction Pembrolizumab is a novel human immunotherapeutic monoclonal antibody directed against PD-1 (programmed death receptor 1). It has been approved by the FDA for treatment of many malignancies including breast cancer. Although it has improved survival for many oncological conditions, it has been linked to a variety of endocrinopathies. We present a case of Pembrolizumab induced hypophysitis with isolated ACTH deficiency presenting as secondary adrenal insufficiency. Case presentation A 36 year old female with a history of breast cancer treated with chemotherapy and surgery, presented with intractable nausea and vomiting. She reported not being able to keep anything down for 4-5 days and had experienced multiple episodes of non-bilious, non-bloody emesis. She endorsed dizziness, but denied fever, chills, shortness of breath, abdominal pain, or problems with urination/bowel movements. She had been treated with Pembrolizumab with the last dose being 4 months prior to presentation. On arrival, vitals included temperature 98.4 F, BP 104/62 mm Hg, Heart rate 104 bpm, RR 16 with SpO2 100%. Labs were significant for WBC count 3.65 K/uL (3.81 - 8.94), Eosinophils 10.4% (0.1 - 6; she was noted to have eosinophilia prior to presentation), Hemoglobin 11.6 g/dL (11 - 14.9), Sodium 140 mmol/L (136-145), Potassium 3.4 mmol/L (3.4 - 5.1), Glucose 57 mg/dL (70 - 100), Lactate 0.7 mmol/L (0.2 -2), Lipase 23 U/L (13 - 60), Urine HCG negative, Urinalysis negative for nitrites and leukocyte esterase, TSH 1.22 uIU. ml (0.5 -5.7) and AM cortisol 0.4 ug/dL (6 - 18.4). Endocrinology was consulted and further work up was sought: ACTH &amp;lt;5 pg/ml (7.2 - 63) and Cortisol 30 - 60 minutes after cosyntropin stimulation 3.6 ug/dL. Prolactin, free T4, FSH and LH were within normal limits. MRI brain showed a prominent pituitary gland with enhancement and interval loss of posterior pituitary bright spot representing lymphocytic hypophysitis. She was started on Solu-Cortef and her symptoms resolved. She was discharged on hydrocortisone with education on sick day rules for secondary adrenal insufficiency. Discussion Adrenal insufficiency secondary to immunotherapy is a rare, emergent and dangerous condition. The key to successful management is early recognition. In patients treated with immunotherapy agents, non-specific symptoms such as nausea, vomiting, fatigue (which are common adverse effects of chemotherapy) should prompt urgent evaluation with hormonal workup. Evidence suggests that eosinophilia may be an early indicator before the onset of symptoms which was evident in our case. Radio-graphic imaging is helpful for diagnosis. Treatment entails high dose corticosteroids for acute crisis followed by physiological maintenance dosing. Patients should be educated on sick day rules for adrenal insufficiency. Presentation: No date and time listed

False positive pregnancy tests in pediatric patients with augmentation enterocystoplasty

Our primary aim was to determine the prevalence of positive over-the-counter (OTC) pregnancy tests among pediatric female and male patients who had undergone augmentation enterocystoplasty. A secondary aim was to determine whether patient factors or urinalysis (UA) results were associated with false positive OTC pregnancy test results in patients with a history of augmentation enterocystoplasty. Patients at a tertiary pediatric hospital who had previously undergone augmentation enterocystoplasty, Mitrofanoff appendicovesicostomy only, and age- and sex-matched controls were prospectively recruited. Urine samples were obtained, and two OTC pregnancy tests were conducted - OTC Test A reported the ability to detect pregnancy at hCG levels as low as 9.6 mIU/mL, while OTC Test B reported positive pregnancy test results at hCG levels ≥25 mIU/mL. A point of care (POC) pregnancy test and UA were also completed. Mann-Whitney U tests and Fisher's exact tests were used to assess whether patient factors or UA results were associated with false positive pregnancy test results. Fifty pediatric patients were enrolled, including 20 with augmentation enterocystoplasty, 20 age- and sex-matched controls, and 10 with Mitrofanoff appendicovesicostomy only. Among the 20 patients who had undergone augmentation cystoplasty, 15 (75.0%) had positive pregnancy test results on OTC Test A, including 11/13 (84.6%) females and 4/7 (57.1%) males. No control patients or patients with Mitrofanoff appendicovesicostomy only had a positive test. No patients had positive pregnancy test results on OTC Test B or the POC test. While patients with augmentation enterocystoplasty were significantly more likely to test positive for blood (p=0.01), nitrate (p=0.03), and leukocytes (p<0.0001), these factors were not significantly associated with false positive pregnancy results. No patient factors or UA results were associated with increased likelihood of false positive OTC pregnancy test results. These findings indicate that sensitive OTC pregnancy tests that detect low quantities of hCG in urine may result in false positive results among patients who have undergone augmentation enterocystoplasty. Female patients of reproductive age with bladder augmentations should be counseled regarding appropriate pregnancy testing options. Additional research is needed to determine the mechanism responsible for false positive pregnancy tests in this patient population.

Recombinant Human Chorionic Gonadotropin Versus Purified Human Chorionic Gonadotropin Trigger for In vitro Fertilization Intracytoplasmic Sperm Injection Cycle

The recombinant human luteinizing hormone or human chorionic gonadotropin (hCG) has substantially taken over the product which was formulated from the urine of pregnant women. In addition to this, a number of randomized controlled trials have been conducted comparing the significance of recombinant hCG (rHCG) with urinary hCG in assisted reproduction. Nevertheless, the present study has collected secondary information based on the nature of the chosen research area. The acquired data and information have been analyzed using content analysis technique to review the methodology and findings of the selected research articles. With the use of rHCG as well as purified hCG, the serum level was equivalent at day 5 among women underwent intracytoplasmic sperm injection (ICSI) but this level abnormally increased among those injected with rHCG thus, minimizing the rate of pregnancy. It has been concluded that purified hCG trigger plays a more efficient role in inducing oocyte maturation and increasing the rate of pregnancy among women undergoing in vitro fertilization ICSI cycle in comparison to rHCG trigger.

P-568 The effect of recombinant hCG on FSH-induced ovarian stimulation in rats depends on the FSH dose and can be detrimental at high concentrations

Abstract Study question What is the effect of choriogonadotropin beta (CG beta) on FSH-induced ovarian stimulation and multifollicular development in a rat model? Summary answer CG beta dose-dependently potentiates effects of low-to-mid FSH doses but has inhibitory effects at high concentrations: optimal CG beta/FSH ratio depends on the FSH dose. What is known already Similarly to follitropin delta (rFSH), CG beta (FE 999302) is a novel recombinant hCG purified from the human PER.C6®cell line. A recent placebo-controlled trial in women undergoing ovarian stimulation with follitropin delta demonstrated that the addition of 1 to 12 µg CG beta reduced the number of intermediate follicles and related hormones. This observation required further preclinical research to (1) evaluate whether the pharmacology of CG beta at LH/CGR was different than other hCG forms used in the clinic and/or (2) assess the effect of high concentrations of hCG and different hCG/FSH ratios on multiple follicular development and follicle atresia. Study design, size, duration Signaling properties of CG beta and other LH/hCG forms were compared at downstream pathways of LH/CGR in recombinant systems and human granulosa cells. To evaluate the effects of FSH±hCG in vivo, juvenile female rats were injected subcutaneously twice daily with follitropin delta ± CG beta/alfa for three days followed by an ovulatory dose of hCG. Oviducts were then collected for oocyte enumeration, ovaries and uteri were weighed, and ovaries were fixed for histological analysis. Participants/materials, setting, methods The pharmacology of CG beta and other LH/hCG forms was evaluated in a cAMP assay in human granulosa cells from follicular fluid from IVF patients and in recombinant systems, at the Gs, Gq and arrestin pathways. In the rat model, a dose response of follitropin delta (Rekovelle) was first evaluated, followed by evaluation of the dose-dependent effects of CG beta (0.00117-2.4 µg/kg), or CG alfa (Ovidrel/Ovitrelle), in combination with 1, 3 or 10 µg/kg rFSH. Main results and the role of chance The in vitro pharmacology (potency and efficacy) of CG beta was similar to recombinant LH, urinary hCG and recombinant hCG (CG alfa) tested at all proximal pathways evaluated downstream of LH/CGR as well as in human granulosa cells. In vivo, treatment with follitropin delta induced a bell-shaped dose-response curve for oocyte release with a maximum response of 40-50 oocytes at 8-10 µg/kg follitropin delta dose. The addition of CG beta dose-dependently potentiated the effects at low-to-mid follitropin delta doses but had inhibitory effects on the number of ovulated oocytes at high CG beta concentrations. The lowest CG beta dose that clearly reduced the number of ovulated oocytes was 2.4, 0.6 and 0.3 µg/kg in combination with a fixed dose of 1, 3 and 10 µg/kg follitropin delta, respectively, which indicated that the optimal hCG/FSH ratio and corresponding hCG efficacious dose was inversely related to the FSH dose. There was no difference between CG beta and CG alfa for the dose effect on the number of ovulated oocytes or ovarian weight. Histology data indicated many cystic follicles following high CG beta exposure which may represent atretic follicles prior to triggering follicular maturation and ovulation. Limitations, reasons for caution This is the first study demonstrating that the FSH dose in combination with the hCG dose determines the effect on multiple follicle growth, ovulation, and atresia. These observations need to be confirmed in clinical research, as doses and ratios applied in the rat cannot be extrapolated to the clinical setting. Wider implications of the findings A better understanding of the effect of different FSH to hCG ratios will help to improve current mixed protocols and design future recombinant combination products providing the optimal treatment outcome for each individual patient. Trial registration number not applicable