P4 Measurements Research Articles

Exogenous estrogen partially rescues progesterone deficiency and autophagosome enlargement in Mcoln1 −/− mouse model with lysosomal storage disorder

Objective: Female Mcoln1 −/− mice exhibit progressive progesterone (P4) deficiency, luteal cell degeneration, and premature embryo implantation failure at five months old. We attempted to rescue embryo implantation in non-virgin Mcoln1 −/− mice (5–6 months old) with exogenous P4 treatment on days 1.5 post-coitum (D1.5), D2.5, and D3.5, and observed partially restored luteal cell morphology on D4.5, but unexpectedly found 17β-estradiol (E2) contamination in the P4 working solution. In this study, we aim to investigate exogenous P4 and/or E2 for the partial recovery of luteal cell morphology in infertile Mcoln1 −/− mice. Methods: Control and non-virgin Mcoln1 −/− mice (5–6 months old) were treated with newly ordered vehicle, P4, E2, or P4+ E2 on D1.5 and D2.5 and dissected on D3.5 for P4 and E2 measurements, ovary histology, immunofluorescence, lipid droplet staining, and transmission electron microscopy. Results: E2 treatment significantly increased serum P4 levels in D3.5 Mcoln1 −/− mice. E2 and P4+E2 treatments, but not P4 treatment alone, largely improved the morphology of D3.5 Mcoln1 −/− corpora lutea, indicated by a more contiguous web-like collagen IV expression pattern, increased heat shock protein 60 expression, and reduced accumulation of large lipid droplets. Transmission electron microscopy revealed extremely enlarged autophagosomes and lipid droplets, lysosomes with lamellar structures, and mitochondria with reduced cristae in vehicle-treated D3.5 Mcoln1 −/− luteal cells, while in E2-treated D3.5 Mcoln1 −/− luteal cells, extremely enlarged autophagosomes and lipid droplets were reduced, indicating improved luteal cell ultrastructure. Conclusion: These findings reveal protective effects of high levels of exogenous E2 on P4 production and lysosomal function in Mcoln1 −/− luteal cells.

P-297 Intensive luteal phase support regimens for endometriosis and adenomyosis patients in hormone replacement therapy frozen embryo transfer cycles – subcutaneous or intramuscular progesterone treatment?

Abstract Study question Does subcutaneous (SC) progesterone treatment provide progesterone levels comparable to intramuscular (IM) progesterone when using a cut-off level of 118nmol/l(37.1ng/ml) during intensive luteal phase support? Summary answer As part of an intensive luteal phase support regimen SC progesterone provides higher P4 levels compared to the use of IM progesterone. What is known already Low serum progesterone (P4) levels in HRT-FET cycles negatively impact reproductive outcomes. This effect is particularly pronounced in patients with endometriosis and adenomyosis, due to reduced progesterone actions and progesterone resistance at the receptor level. Therefore, higher serum P4 levels are required to maintain an optimal reproductive outcome during HRT-FET. A P4 cut-off of 118nmol/l (37.1ng/ml) or above has been suggested, a level four times higher than that of the non-endometriosis patient. To achieve high P4 levels, intensive luteal phase support (LPS) regimen has been suggested. This includes vaginal progesterone administration and additional treatment via IM, SC or rectal administration. Study design, size, duration This cohort study includes 380 HRT-FET cycles in endometriosis/adenomyosis patients. Patients were treated from January 2016 until August 2019 with one type of intensive LPS regimen (n = 262) and from January 2000 to January 2023 with two different LPS regimens depending on whether serum P4 levels were lower or higher than 118nmol/l on the day of blastocyst transfer (BT) (n = 118). P4 was measured in a standardised manner. Participants/materials, setting, methods Patients received treatment in a public university affiliated fertility clinic. Endometrial priming involved the use of estradiol followed by vaginal progesterone. From the fourth day of progesterone, one of the following intensive LPS regimens were added: A) 50mg IM progesterone, B) 25mg SC progesterone BD, or C) 25mg SC progesterone BD plus 400mg rectal rescue progesterone BD if P4<118nmol/l. Blastocyst transfer (BT) and P4 measurement were performed on the sixth day of progesterone administration. Main results and the role of chance The overall pregnancy rate, live birth rate (LBR) and total pregnancy loss rates for the whole cohort were 63%, 41% and 36%, respectively. For the three groups A, B and C the mean serum P4 levels on the day of BT were 103.1 ±44.4nmol/l, 170.5 ±62.9nmol/l and 90.1 ±21.5nmol/l. In Group A (IM) 67% of patients (176/262) had P4 levels <118 nmol/l whereas only 36% (43/118; p < 0.001) had P4 <118 nmol/l in the groups of patients treated with SC progesterone (Group B and C). The unadjusted LBR was not significantly different between groups, 39%, 39%, 51%, (p = 0.32). However, a sub-group analysis showed that comparing patients with serum P4 levels <118nmol/l on the BT day in Group A with patients with P4 levels <118nmol/l in Group C a significantly higher LBR was found if intensive LPS included both SC and rectally administered progesterone compared to IM progesterone, only: 51% (44/86) vs 34% (59/176), p = 0.03. The adjusted odds ratio for live birth was 2.04 (95% CI [1.00, 4.16], p = 0.05) after adjusting for age, vitrification day 5 or 6, blastocyst quality and numbers of blastocysts transferred, if intensive LPS included SC and rectal rescue compared to IM, only in patients with P4 levels <118nmol/l. Limitations, reasons for caution This cohort study was conducted over two different time periods. Single embryo transfer was performed only during the later period. The bio-pharmacological profiles of SC and IM progesterone differ, which can influence serum P4 levels. However, in the present study, all blood samples were collected in a standardised manner. Wider implications of the findings This study is the first to report reproductive outcomes following different intensive LPS regimens in a cohort of endometriosis/adenomyosis patients. To increase reproductive outcomes, individualisation of the HRT-FET cycle may be beneficial and, importantly, rectal progesterone administration can be used as a rescue regimen in addition to SC progesterone. Trial registration number Due to the retrospective study design no ethical approval was needed.

P-599 Oestradiol/Progesterone ratio Impact on Frozen Embryo Transfer (FET) and pregnancy outcomes

Abstract Study question Are oestradiol (E2) levels and oestradiol/progesterone (P4) ratio related to higher implantation rates and reduced risk of miscarriage? Summary answer We concluded that higher E2 levels and an increased E2/P4 ratio on the day before FET were associated with lower implantation rates. What is known already A progesterone threshold < 9.2 ng/ml on the day of FET has been related to lower rates of clinical pregnancy. Progesterone is responsible for increasing endometrial vascularization and exerting an immunomodulator effect that allows blastocyst implantation. How oestradiol affects implantation is not well described yet. An appropriate luteal phase support is essential to synchronize the endometrium, ensure proper implantation and reduce the risk of abortion. However, no studies exist regarding E2 levels and E2/P4 ratios related to pregnancy outcomes in the first weeks of pregnancy. Study design, size, duration This is a prospective cohort study. From February 2023 to July 2023, 35 patients who underwent IVF in a private ART medical center were enrolled. Only euploid embryos after PGT- A were transferred and only the first single FET per patient was included. All embryos were transferred in blastocyst stage. Mosaic embryos, fresh cycles, patients with no PGT-A, miscarriages beyond 10 weeks and chemical pregnancies (no evidence of gestational sac) were excluded. Participants/materials, setting, methods 35 patients who underwent IVF-ICSI were retrospectively analyzed. Serum oestradiol, serum progesterone and E2/P4 ratios were measured the day before embryo transfer and later in three moments: 15 days after positive BHCG test, when gestational sac was visualized; 10-15 days after visualization of the gestational sac, when positive embryo heart rate was verified, and in any moment of vaginal bleeding until the 10th week of pregnancy. Main results and the role of chance Our study supported the hypothesis that E2/P4 ratio before FET may be an important factor to predict the risk of threatened abortion considering that all patients had P4 levels > 10 ng/ml before FET. Multiple logistic regression revealed that E2 and E2/p4 ratio on the day before FET are variables related to the risk of threat of abortion. The resulting adjusted OR for E2 on the day before FET in patients who suffered a threatened abortion vs patients who did not experience threat of abortion was 1,025 (95% CI: 1,001-1,049), p value 0,041. The OR for E2/p4 ratio before FET in patients with treat of abortion vs those without threat of abortion was 1,235 (95% CI: 1,035-1,472), p value 0,019. ROC curves showed a significant predictive value for E2 levels >220 pg/ml [AUC of 0,88 (95% CI: 0,73-1,0), p value 0,009] and E2/P4 ratio > 23 [AUC 0,91 (95% CI: 0,78-1,0), p < 0,05], the day before FET indicating a higher risk of threat of miscarriage and loss of pregnancy. Multiple logistic regression did not find significant statistical differences, either E2 or E2/P4 ratio, between the two groups of patients in subsequent determinations at 4 and 6 weeks after transfer. Limitations, reasons for caution The main limitation of this study is the sample size with the lack of further serial measurements of oestradiol and p4 in patients that presented threat of abortion/ miscarriage before the 10th week of pregnancy. Wider implications of the findings E2/P4 ratio the day before FET seems to be the most important factor to decrease threat of abortion in patients with P4> 10 ng/ml. Our study may hold predictive value regarding endometrial receptivity and provide clinicians a guide to individualise FET protocols and to prevent early pregnancy losses Trial registration number N/A

P-429 Measuring serum progesterone levels on the day of a fresh blastocyst transfer is debatable as there is no evidence for correlation with ongoing pregnancy results

Abstract Study question Does serum progesterone (P4) level on the day of a fresh blastocyst transfer relate to ongoing pregnancy rate (OPR)? Summary answer OPR after a fresh single blastocyst transfer seems not to be associated with serum P4 level on the day of transfer. What is known already Several studies have suggested an optimal serum P4 threshold for embryo transfers in frozen embryo transfer cycles. However, for fresh embryo transfers, data are scarce and conflicting. Study design, size, duration A retrospective single-center cohort study was performed on 598 complete data cycles, retained out of 631 consecutive fresh single blastocyst transfer cycles. Data were collected from patients undergoing a fresh IVF/ICSI cycle with autologous oocytes at a tertiary fertility center between June 2022 and August 2023. Participants/materials, setting, methods A total of 482 unique patients underwent one or more single fresh blastocyst transfers using micronized vaginal progesterone (MVP) 800mg BID for luteal phase support starting day after oocyte retrieval. Serum P4 levels were determined on day of transfer. The primary endpoint was OPR, positive hCG-test and early pregnancy loss (EPL) were secondary endpoints. Unadjusted and adjusted generalized estimating equations (GEE) assessed P4 level association with the endpoints, using an exchangeable working correlation matrix. Main results and the role of chance No association was identified between P4 levels on day of transfer and OPR after accounting for maternal age, blastocyst quality, P4 and estradiol level at last ultrasound, BMI and endometrial thickness. Data were grouped according to 10/50/90 and 25/50/75 percentiles, resulting in groups < = or > 27ng/ml and < = or > 40ng/ml, with omission of 90th and 75th percentile groups due to right-skewed distribution. With serum P4 levels higher than 40ng/ml, the probability of a positive hCG-test significantly increased (OR 1.79, CI 1.11-2.91; p = 0.018). No significant difference was observed in patients with serum P4 < = or > 27ng/ml (OR 1.23, CI 0.66-2.30; p = 0.506). Regarding OPR, no significant differences were observed when comparing patients with serum P4 < = or > 27/ng/ml or < = or > 40ng/ml (OR 0.88, CI 0.42-18.85; p = 0.736 and OR 1.41, CI 0.83-2.37; p = 0.203, respectively). Moreover, EPL did not significantly differ between serum P4 level groups (OR 1.93, CI 0.61-6.13; p = 0.265 and OR 1.2, CI 0.51-2.84; p = 0.674). Age (p < 0.001) and embryo quality (p < 0.001) are significantly related with OPR. Additionally, late follicular progesterone levels and OPR are significantly associated (OR 2.39, CI 1.16-4.95 in patients with P4 < = or > 27/ng/ml and OR 2.21, CI 1.07-4.55 in patients with < =40ng/ml and >40ng/ml). Limitations, reasons for caution Limitations include the retrospective design and the lack of live birth outcome data. Progesterone levels exceeding 60 ng/ml were not further analyzed to their precise value but grouped as 60 plus which did not allow to identify potential detrimental effects of very high P4 levels on treatment outcome. Wider implications of the findings Our data suggest that measuring P4 on the day of a single blastocyst transfer with MVP luteal phase support may not be indicative of OPR or the risk of EPL. This raises questions regarding the necessity for routine P4 measurements as well as the development of rescue strategies. Trial registration number ONZ-2023-0628

A nitrogen-doped hollow carbon nanospheres-based aptasensor for non-invasive salivary detection of progesterone

Developing an easy-to-use and non-invasive sensor for monitoring progesterone (P4) as a multi-functional hormone is highly demanded for point-of-care testing. In this study, an ultrasensitive electrochemical aptasensor is fabricated for monitoring P4 in human biofluids. The sensing interface was designed based on the porous nitrogen-doped hollow carbon spheres (N-HCSs). The N-HCSs covalently immobilized high-dense aptamer (Apt) sequences as the bioreceptor of P4. The electron transfer of the redox probe was hindered by incubating P4 on the aptasensor surface and forming the P4-Apt complexes. Meanwhile, the signaling was decreased under two wide linear dynamic ranges (LDRs) from 10 fM to 5.6 μM with a limit of detection (LOD) value of 3.33 fM. The aptasensor presented satisfactory selectivity in the presence of different off-target species with successful feasibility for P4 detection in some human urine and saliva samples. The aptasensor with high sensitivity, as an advantage for on-site and sensitive measurement of P4, can be considered a non-invasive tool for routine analysis of real-world clinical samples method.

The combination of dydrogesterone and micronized vaginal progesterone can render serum progesterone level measurements on the day of embryo transfer and rescue attempts unnecessary in an HRT FET cycle.

To evaluate the role of serum progesterone (P4) on the day of embryo transfer (ET) when dydrogesterone (DYD) and micronized vaginal progesterone (MVP) are combined as luteal phase support (LPS) in a hormone replacement therapy (HRT) frozen ET (FET) cycles. Retrospective study, including single euploid HRT FET cycles with DYD and MVP as LPS and P4 measurement on ET day. Initially, patients with P4 levels < 10 ng/ml increased MVP to 400 mg/day; this "rescue" was abandoned later. 560 cycles of 507 couples were included. In 275 women, serum P4 level was < 10 ng/ml on the ET day. Among those with low P4 levels, MVP dose remained unchanged in 65 women (11.6%) and was increased in 210 women (37.5%). Women with P4 levels ≥ 10 ng/ml continued LPS without modification. Overall pregnancy rates in these groups were 61.5% (40/65), 54.8% (115/210), and 48.4% (138/285), respectively (p = n.s.). Association of serum P4 levels with ongoing pregnancy rates was analyzed in women without any additional MVP regardless of serum P4 levels (n = 350); multivariable analysis (adjusted for age, BMI, embryo quality (EQ)) did not show a significant association of serum P4 levels with OPR (OR 0.96, 95% CI 0.90-1.02; p = 0.185). Using inverse probability treatment weights, regression analysis in the weighted sample showed no significant association between P4 treatment groups and OP. Compared to fair EQ, the transfer of good EQ increased (OR 1.61, 95% CI 1.22-2.15; p = 0.001) and the transfer of a poor EQ decreased the odds of OP (OR 0.73, 95% CI 0.55-0.97; p = 0.029). In HRT FET cycle, using LPS with 300 mg/day MVP and 30 mg/day DYD, it appears that serum P4 measurement and increase of MVP in patients with P4 < 10 ng/ml are not necessary.

Associations of postpartum health with progesterone after insemination and endocrine signaling during early pregnancy in dairy cows

Our objective was to investigate associations of postpartum health with serum progesterone (P4) concentrations after first artificial insemination (AI), expression of interferon-stimulated genes (ISG) in blood leukocytes, and serum pregnancy-associated glycoprotein (PAG) during early pregnancy in dairy cows. In this observational study, Holstein cows (n = 475) from 2 commercial herds were enrolled 3 wk before expected parturition. Body condition score (BCS) and lameness were assessed at enrollment, and total Ca, haptoglobin, and nonesterified fatty acids (NEFA) were measured in serum at 2 and 6 ± 2 d in milk (DIM). Blood β-hydroxybutyrate and metritis were evaluated at 4, 8, 11, and 15 ± 2 DIM, and purulent vaginal discharge (PVD) and endometritis based on endometrial cytology were diagnosed at 35 DIM. Onset of cyclicity was assessed by biweekly P4 measurements. The BCS was assessed at enrollment and at 63 DIM, and lameness at enrollment and at 21 and 49 DIM. First AI was based on estrus detection until ∼75 DIM, or synchronization for timed AI thereafter. Serum P4 was measured at d 8 and 12 after first AI; expression of ISG (ISG15 and RTP4) in blood leukocytes was assessed at d 19; and PAG in serum of pregnant cows was measured at d 29, 33, and 40. Multivariable linear regression models were built including health variables (with metabolites categorized via receiver operating characteristic curve analysis) and covariates (season, milk yield, AI method, DIM), accounting for pregnancy status (for ISG and PAG models), repeated measures (for P4 and PAG models), and herd as random. Results are reported comparing cows affected versus unaffected by each predictor. Serum P4 concentrations were lesser at d 8 after AI in cows that had total Ca ≤2.09 mM (3.6 vs. 4.0 ± 0.2 ng/mL) at 2 DIM or a clinical disease (3.7 vs. 4.0 ± 0.2 mg/mL), and P4 tended to be lesser at d 8 in cows that had PVD (3.6 vs. 4.0 ± 0.2 ng/mL) or any uterine disease (3.7 vs. 4.1 ± 0.2 ng/mL). Among cows pregnant at d 29, relative ISG15 expression (fold change) at d 19 was greater in cows that had lameness (5.10 [4.18-6.18] vs. 3.14 [3.10-3.19]) but tended to be lesser in cows that had endometritis (3.20 [2.89-3.54] vs. 4.29 [4.20-4.35]), and relative expression of RTP4 tended to be lesser in cows that had displaced abomasum (1.02 [0.62-1.67] vs. 1.75 [1.73-1.78]). Serum PAG was lesser at d 29 (3.8 vs. 4.2 ± 0.2 sample-to-positive ratio [S:P]) in cows that had NEFA ≥0.73 mM at 6 DIM, and lesser at d 33 (3.9 vs. 4.5 ± 0.2 S:P) and 40 (3.1 vs. 3.7 ± 0.2 S:P) in cows that had prepartum BCS ≥3.75. These findings suggest long-term effects of metabolic or reproductive tract disorders on luteal function after first AI, pregnancy recognition signaling, and placental function during early pregnancy. These associations may partially explain some of the pathways linking postpartum health and fertility in dairy cows.

Rectal progesterone administration secures a high ongoing pregnancy rate in a personalized Hormone Replacement Therapy Frozen Embryo Transfer (HRT-FET) protocol: a prospective interventional study.

Can supplementation with rectal administration of progesterone secure high ongoing pregnancy rates (OPRs) in patients with low serum progesterone (P4) on the day of blastocyst transfer (ET)? Rectally administered progesterone commencing on the ET day secures high OPRs in patients with serum P4 levels below 35 nmol/l (11 ng/ml). Low serum P4 levels at peri-implantation in Hormone Replacement Therapy Frozen Embryo Transfer (HRT-FET) cycles impact reproductive outcomes negatively. However, studies have shown that patients with low P4 after a standard vaginal progesterone treatment can obtain live birth rates (LBRs) comparable to patients with optimal P4 levels if they receive additionalsubcutaneous progesterone, starting around the day of blastocyst transfer. In contrast, increasing vaginal progesterone supplementation in low serum P4 patients does not increase LBR. Another route of administration rarely used in ART is the rectal route, despite the fact that progesterone is well absorbed and serum P4 levels reach a maximum level after ∼2 h. This prospective interventional study included a cohort of 488 HRT-FET cycles, in which a total of 374 patients had serum P4 levels ≥35 nmol/l (11 ng/ml) at ET, and 114 patients had serum P4 levels <35 nmol/l (11 ng/ml). The study was conducted from January 2020 to November 2022. Patients underwent HRT-FET in a public Fertility Clinic, and endometrial preparation included oral oestradiol (6 mg/24 h), followed by vaginal micronized progesterone, 400 mg/12 h. Blastocyst transfer and P4 measurements were performed on the sixth day of progesterone administration. In patients with serum P4 <35 nmol/l (11 ng/ml), 'rescue' was performed by rectal administration of progesterone (400 mg/12 h) starting that same day. In pregnant patients, rectal administration continued until Week 8 of gestation, and oestradiol and vaginal progesterone treatment continued until Week 10 of gestation. Among 488 HRT-FET single blastocyst transfers, the mean age of the patients at oocyte retrieval (OR) was 30.9 ± 4.6 years and the mean BMI at ET 25.1 ± 3.5 kg/m2. The mean serum P4 level after vaginal progesterone administration on the day of ET was 48.9 ± 21.0 nmol/l (15.4 ± 6.6 ng/ml), and a total of 23% (114/488) of the patients had a serum P4 level lower than 35 nmol/l (11 ng/ml). The overall, positive hCG rate, clinical pregnancy rate, OPR week 12, and total pregnancy loss rate were 66% (320/488), 54% (265/488), 45% (221/488), and 31% (99/320), respectively. There was no significant difference in either OPR week 12 or total pregnancy loss rate between patients with P4 ≥35 nmol/l (11 ng/ml) and patients with P4 <35 nmol/l, who received rescue in terms of rectally administered progesterone, 45% versus 46%, P = 0.77 and 30% versus 34%, P = 0.53, respectively. OPR did not differ whether patients had initially low P4 and rectal rescue or were above the P4 cut-off. Logistic regression analysis showed that only age at OR and blastocyst scoring correlated with OPR week 12, independently of other factors like BMI and vitrification day of blastocysts (Day 5 or 6). In this study, vaginal micronized progesterone pessaries, a solid pessary with progesterone suspended in vegetable hard fat, were used vaginally as well as rectally. It is unknown whether other vaginal progesterone products, such as capsules, gel, or tablet, could be used rectally with the same rescue effect. A substantial part of HRT-FET patients receiving vaginal progesterone treatment has lowserum P4. Adding rectally administered progesterone in these patients increases the reproductive outcome. Importantly, rectal progesterone administration is considered convenient, and progesterone pessaries are easy to administer rectally and of low cost. Gedeon Richter Nordic supported the study with an unrestricted grant as well as study medication. B.A. has received unrestricted grant from Gedeon Richter Nordic and Merck and honoraria for lectures from Gedeon Richter, Merck, IBSA and Marckyrl Pharma. P.H. has received honoraria for lectures from Gedeon Richter, Merck, IBSA and U.S.K. has received grant from Gedeon Richter Nordic, IBSA and Merck for studies outside this work and honoraria for teaching from Merck and Thillotts Pharma AB and conference expenses covered by Merck. The other co-authors have no conflict of interest to declare. EudraCT no.: 2019-001539-29.

Validation of Immulite 2000XPI chemiluminescent immunoassay for cattle serum progesterone measurement

Monitoring circulating progesterone concentration (P4) is an important component of basic and applied reproduction re­search and clinical settings. Radioimmunoassay (RIA) is con­sidered the gold standard method for measuring P4 in cattle blood, but it generates radioactive waste, has low throughput, and requires licensed facilities. Immulite® 2000XPI (Siemens) is a fully automated immunoassay system marketed for human use to measure concentrations of different analytes including P4. Our objectives were therefore to: 1) validate cattle serum P4 measurement with the Immulite 2000XPI; 2) characterize the analytical performance of the Immulite 2000XPI P4 immunoas­say (IPI) across the reportable concentration range.

Validation of Immulite® 2000XPI chemiluminescent immunoassay for sheep serum progesterone measurement

Monitoring circulating progesterone concentration (P4) is an important component of basic and applied reproduction re­search and clinical settings. Radioimmunoassay (RIA) is con­sidered the gold standard method for measuring P4 in sheep blood, but it generates radioactive waste, has low throughput and requires licensed facilities. Immulite® 2000XPI (Siemens) is a fully automated immunoassay system marketed for human use to measure concentrations of different analytes including P4. Our objectives were therefore to: 1) validate sheep serum P4 measurement with the Immulite 2000XPI; and 2) characterize the analytical performance of the Immulite 2000XPI P4 immu­noassay (IPI) across the reportable concentration range.

P-596 Rectal progesterone administration secures high ongoing pregnancy rate in a personalized Hormone Replacement Therapy Frozen Embryo Transfer (HRT-FET) protocol - a prospective interventional study

Abstract Study question Can supplementation with additional rectal administration of progesterone secure high ongoing pregnancy rates (OPR) in patients with low serum progesterone (P4) at blastocyst transfer (ET)? Summary answer Rectally administered progesterone starting on the blastocyst transfer day secures high OPR in patients with serum P4 levels lower than 35 nmol/l (11ng/ml). What is known already Low serum P4 levels at peri-implantation in HRT-FET cycles impact reproductive outcomes negatively. However, studies have shown that patients with low P4 after a standard vaginal progesterone treatment can obtain live birth rates (LBR) comparable to patients with optimal P4 levels if they receive additional subcutaneous progesterone, starting close to the day of blastocyst transfer. In contrast, increasing vaginal progesterone supplementation in low serum P4 patients does not increase LBR. Another route of administration rarely used in ART is the rectal route, although progesterone is well absorbed and serum P4 levels reach a maximum level after approximately two hours. Study design, size, duration This prospective interventional study included a cohort of 488 patients treated with single blastocyst HRT-FET, in which a total of 374 patients had a serum P4 level ≥35nmol/l (11ng/ml) at ET, and 114 patients had a serum P4 level &amp;lt;35nmol/l (11ng/ml). The study was conducted from January 2020 to November 2022. Participants/materials, setting, methods Patients undergoing HRT-FET in a public Fertility Clinic. Endometrial preparation included oral oestradiol (6mg/24hours), followed by vaginal micronized progesterone, 400mg/12hours. Single blastocyst transfer and P4 measurements were performed on the 6th day of progesterone administration and patients with serum P4 &amp;lt;35nmol/l (11ng/ml) additional rectal administration of progesterone (400mg/12hour) was started the same day. In pregnant patients, vaginal and rectal administration continued until week 8, and oestradiol and vaginal progesterone treatment continued until week 10. Main results and the role of chance Among 488 HRT-FET single blastocyst transfers, the mean age of the patients at oocyte retrieval (OR) was 30.9 ±4.6 years and the mean BMI at ET 25.1 ±3.5 kg/m2. The mean serum P4 level after vaginal progesterone administration on the day of blastocyst transfer was 48.9 ±21.0 nmol/l (15.4 ±6.6ng/ml), and a total of 23% (114/488) of the patients had a serum P4 level lower than 35nmol/l (11ng/ml). The overall, positive hCG rate, clinical pregnancy rate, OPR week 12, and total pregnancy loss rate were 66% (320/488), 54% (265/488), 45% (221/488), and 31% (99/320), respectively. There was no significant difference in neither OPR week 12 nor total pregnancy loss rate between patients with P4 ≥35nmol/l (11ng/ml) and patients with P4 &amp;lt;35nmol/l, who received rescue in terms of rectally administered progesterone, 45% vs. 46%, p = 0.77 and 30% vs. 34%, p = 0.53, respectively. OPR did not depend on whether patients had initially low P4 and rectal rescue or were above the P4 cut-off. Logistic regression analysis showed that only age at OR and blastocyst scoring correlated with OPR week 12, independently of other factors like BMI, and vitrification day of blastocysts (day 5 or 6). Limitations, reasons for caution In this study vaginal progesterone (Cyclogest®), a solid pessary with progesterone suspended in vegetable hard fat, was used vaginally as well as rectally. It is unknown whether other vaginal progesterone products could be used rectally as easily with the same rescue effect. Wider implications of the findings A substantial part of HRT-FET patients receiving vaginal progesterone treatment has low serum P4. Adding rectally administered progesterone in these patients increases the reproductive outcome. Importantly, rectal progesterone administration is considered convenient by the majority of patients; moreover, progesterone pessaries are easy to administer rectally and of low cost. Trial registration number EudraCT no.: 2019-001539-29

P-607 Interindividual variation of progesterone elevation post LH rise: Implications for natural cycle frozen embryo transfers in the individualized medicine era.

Abstract Study question To what extent does the period between LH-rise and P4-rise vary in ovulatory natural menstrual cycles? Summary answer Three distinct ovulatory patterns were noted defined as the period between LH-rise and P4-rise and varied by up to two days. What is known already The key to success of FET in a NC is the accurate diagnosis of ovulation to determine the optimal timing of embryo transfer. The most common method used to determine time of ovulation in NC is the detection of luteinizing hormone (LH) surge. It is well recognized that the endocrine profile of menstrual cycles varies not only amongst women but also from cycle to cycle for any given female. Most hormone trajectories in individual women differ considerably from the mean hormonal curves and LH surges culminating in ovulation appear to be highly variable in timing, amplitude and duration. Study design, size, duration Retrospective, observational study including 102 women who underwent endocrine monitoring during a frozen embryo transfer in a natural cycle between 1st January 2017 and 31st August 2021 in a tertiary IVF centre. Participants/materials, setting, methods Ultrasound scans were performed to monitor follicular growth. Serial measurements of serum LH, FSH, estradiol and P4 were commenced once a dominant follicle measuring ≥ 14mm was identified and on three consecutive days until (including) the day of ovulation. The LH surge was considered to have begun when the concentration rose by 180% above the most recent serum value. Progesterone concentrations of 1.0ng/ml and above were regarded as confirmation of ovulation to schedule FET. Main results and the role of chance Patients’ characteristics presented as median and (range): age 35 years (23-43), BMI 25.24kg/m2 (17.32-38.21), AMH 2.07 ng/ml (0.06-8.12). Twenty-one (20.6%) women had the LH-rise 2 days prior to P4 -rise, 71 (69.6%) had on the day immediately preceding and 10 (9.8%) on the same day of P4-rise. If FET was scheduled based on LH-rise, 30.4% of the patients would have their FET scheduled on a different day than FET scheduled according to P4-rise. The period between LH-rise and P4-rise would be approximately one day longer in 20.6% of the participants and one day shorter in 9.8% than the anticipated 24 hour period. There was a significant difference in body mass index between those women who had LH-rise 2 days prior to P4-rise (BMI 26.91 kg/m2 (range 23.74-30.75) and the women who had LH-rise on the same day (BMI 22.39 kg/m2 (range 20.91-24.96) (p = 0.03). The AMH levels were also found to be significantly lower in those women who had LH rise 2 days prior to P4-rise ( AMH 1.59 ng/ml (range: 0.82-2.09) as compared to those who had LH-rise on the same day (AMH 2.67ng/ml (range: 1.83—6.63) (p = 0.04). LH may not serve as the best benchmark to schedule FET. Limitations, reasons for caution Frozen embryo transfers were scheduled according to serum progesterone levels therefore our study does not provide a direct answer to the question whether FET scheduled according to serum progesterone levels provides higher live birth rates than FET scheduled based on urinary or serum LH levels in a natural cycle. Wider implications of the findings Variation in the period between LH rise and progesterone rise in ovulatory cycles has implications for the choice of marker for the start of secretory transformation in frozen embryo transfer cycles. The study participants are representative of the relevant population of women undergoing frozen embryo transfer in a natural cycle. Trial registration number Not Applicable

P-595 What to expect from “standard vaginal progesterone regimen” in Hormone Replacement Therapy Frozen Embryo Transfer (HRT-FET) – do different products result in different serum levels?

Abstract Study question Do luteal serum progesterone levels (P4) vary in relation to time of blood sampling, the type and dosing of vaginal progesterone products used for HRT-FET? Summary answer Significant differences in luteal serum P4 levels exist between time of blood sampling as well as between different vaginal progesterone products and the administration regimen. What is known already Mid-luteal P4 levels below 9-11 ng/ml have been shown to negatively affect reproductive outcomes in HRT-FET cycles. Formulations of vaginal progesterone products differ as progesterone can be suspended in vegetable hard fat, in a vegetable lipophile liquor, be prepared in an oil-in-water emulsion carrier, or mixed in a tablet. Moreover, dosing regimens vary from one to three times daily, and BMI, age, and parity affect serum P4 levels; importantly, no consensus exists in terms of optimal time point to measure P4 in relation to administration, the specific serum P4 cut-off level to use or the most optimal vaginal progesterone product. Study design, size, duration Cohort study including 488 HRT-FET blastocyst transfers performed from January 2020 to November 2022 in patients undergoing a “standard vaginal progesterone regimen”. Each patient participated only once in the study. To compare the vaginal progesterone product used in the present study, we reviewed the most recent literature, including data from six studies, using three different products in six different regimens; all studies were cohort studies, including between 227 -1150 patients undergoing a “standard” HRT-FET protocol. Participants/materials, setting, methods A total of 488 patients in a public Fertility Clinic underwent HRT-FET, including endometrial preparation with oral oestradiol (6mg/24hours), followed by vaginal micronized progesterone, 400mg/12hours (Cyclogest®). Blood sampling on the blastocyst transfer day was standardised to two to four hours after progesterone administration. In contrast, P4 was measured randomly on the day of pregnancy testing. For comparison a review of the literature from six HRT-FET cohort studies using either Crinone®, Utrogestan® or “PIVET”-pessaries was performed. Main results and the role of chance The mean serum P4 (2-4 hours after administration) on the day of blastocyst transfer in our cohort was 15.4 ±6.6ng/ml. On the day of pregnancy testing (random time points) serum P4 levels were divided into four groups, depending on the time from progesterone administration to blood sampling: &amp;lt;2hours, &amp;gt;2&amp;lt;4hours &amp;gt;4&amp;lt;6hours and &amp;gt;6hours.The mean P4 levels were 14.7 ±6.3ng/ml, 15.6 ±5.8ng/ml, 14.3 ±4.9ng/ml and 12.9 ±6.4, respectively, and a significant difference was seen, between P4 levels measured 2-4 hours and &amp;gt;6 hours after vaginal administration (p = 0.03). As regards the product, the mean P4 levels in this study (Cyclogest®) was significantly different from the reviewed P4 levels of other “standard” HRT-FET cohorts, using three different products (Uterogestan®, Crinone® and the “PIVET-pessary”) 12.1 ±7.0ng/ml (p &amp;lt; 0.001), 7.6 ±3.2ng/ml (p &amp;lt; 0.001) and 29.3 ±20.3ng/ml (p &amp;lt; 0.001), respectively. As regards the dose, a significant difference in P4 levels was seen between dosing regimens in two cohorts when using the same product (Crinone®) 90mg x 3 vs. 90 mg x 2; 11.3 ±4.7ng/ml and 7.6 ±3.2ng/ml, respectively, p &amp;lt; 0.001. Interestingly, when comparing another product (Utrogestan®) used in different dosing regimens, 400mg x 2 vs. 200mg x 3, no difference in P4 levels was seen, 12.1 ±7.0ng/ml and 11.3 ±5.1ng/ml, P = 0.09. Limitations, reasons for caution In HRT-FET serum P4 levels reflect the exogenous administration, only, and P4 fluctuations depend on the route and number of administrations, as well as absorption and metabolization. The reviewed data used for this comparison between different progesterone products and regimens derive from cohorts with different characteristics and blood sampling timings. Wider implications of the findings Luteal serum P4 measurement is important, however, standardization of blood sampling in relation to the latest vaginal progesterone administration is mandatory to obtain correct levels. Clinicians should be aware of the significant differences in P4 levels related to dose and vaginal absorption between different types of vaginal progesterone products. Trial registration number EudraCT no.: 2019-001539-29

Application of Enzyme-Linked Fluorescence Assay (ELFA) to Obtain In Vivo Matured Dog Oocytes through the Assessment of Progesterone Level.

Successful dog cloning requires a sufficient number of in vivo matured oocytes as recipient oocytes for reconstructing embryos. The accurate prediction of the ovulation day in estrus bitches is critical for collecting mature oocytes. Traditionally, a specific serum progesterone (P4) range in the radioimmunoassay (RIA) system has been used for the prediction of ovulation. In this study, we investigated the use of an enzyme-linked fluorescence assay (ELFA) system for the measurement of P4. Serum samples of estrus bitches were analyzed using both RIA and ELFA, and the measured P4 values of ELFA were sorted into 11 groups based on the standard concentration measured in RIA and compared. In addition, to examine the tendency of changes in the P4 values in each system, the P4 values on ovulation day (from D - 6 to D + 1) in both systems were compared. The ELFA range of 5.0-12.0 ng/mL was derived from the RIA standard range of 4.0-8.0 ng/mL. The rates of acquired matured oocytes in RIA and ELFA were 55.47% and 65.19%, respectively. The ELFA system successfully produced cloned puppies after the transfer of the reconstructed cloned oocytes. Our findings suggest that the ELFA system is suitable for obtaining in vivo matured oocytes for dog cloning.

Are systemic progesterone levels in true natural cycle euploid frozen embryo transfers with luteal phase support predictive for ongoing pregnancy rates?

Are serum progesterone (P4) levels on the embryo transfer (ET) day predictive of ongoing pregnancy (OP) following a single euploid blastocyst transfer in a natural cycle (NC) when luteal phase support is routinely given? In single euploid frozen ETs in NC, P4 levels on ET day are not predictive for OP, when luteal phase support (LPS) is routinely added after the ET. In an NC frozen embryo transfer (FET), P4 produced by the corpus luteum initiates secretory transformation of the endometrium and maintains pregnancy after implantation. There are ongoing controversies on the existence of a P4 cutoff level on the ET day, being predictive for the chance of OP as well as of the possible role of additional LPS after ET. Previous studies in NC FET cycles, evaluating and identifying P4 cutoff levels did not exclude embryo aneuploidy as a possible reason for failure. This retrospective study analyzed single, euploid FET in NC, conducted in a tertiary referral IVF centre between September 2019 and June 2022, for which measurement of P4 on the day of ET and the treatment outcomes were available. Patients were only included once into the analysis. Outcome was defined as OP (ongoing clinical pregnancy with heartbeat, >12 weeks) or no-OP (not pregnant, biochemical pregnancy, early miscarriage). Patients with an ovulatory cycle and a single euploid blastocyst in an NC FET cycle were included. Cycles were monitored by ultrasound and repeated measurement of serum LH, estradiol, and P4. LH surge was identified when a rise of 180% above the previous level occurred and P4 levels of ≥1.0 ng/ml were regarded as confirmation of ovulation. The ET was scheduled on the fifth day after P4 rise and vaginal micronized P4 was started on the day of ET after P4 measurement. Of 266 patients included, 159 (59.8%) patients had an OP. There was no significant difference between the OP- and no-OP-groups for age, BMI, and day of embryo biopsy/cryopreservation (Day 5 versus Day 6). Furthermore, P4 levels were not different between the groups of patients with OP (P4: 14.8 ng/ml (IQR: 12.0-18.5 ng/ml)) versus no-OP (P4: 16.0 ng/ml (IQR: 11.6-18.9 ng/ml)) (P = 0.483), and no differences between both groups, when P4 levels were stratified into categories of P4 levels of >5 to ≤10, >10 to ≤15, >15 to ≤20, and >20 ng/ml (P = 0.341). However, both groups were significantly different for the embryo quality (EQ), defined by inner cell mass/trophectoderm, as well as when stratified into three EQ groups (good, fair, and poor) (P = 0.001 and 0.002, respectively). Stratified EQ groups remained the only significant parameter influencing OP in the uni- and multivariate analyses (P = 0.002 and P = 0.004, respectively), including age, BMI, and P4 levels (each in categories) and embryo cryopreservation day. Receiver operator characteristic curve for the prediction of an OP revealed an AUC of 0.648 when age, BMI and EQ groups were included into the model. The inclusion of P4 measurement on ET day into the model did not add any benefit for OP prediction (AUC = 0.665). The retrospective design is a limitation. Monitoring serum P4 levels can be abandoned in NC FET cycles with routine LPS as they do not seem to be predictive of live birth. No external funding was used for this study. The authors state that they do not have any conflicts of interest. N/A.

The effect of rifampin on serum etonogestrel concentrations and biomarkers of ovulation among contraceptive implant users: A pharmacokinetic and pharmacodynamic study

ObjectivesRifampin, a strong CYP3A inducer, is the gold standard for evaluating CYP3A-mediated drug-drug interactions. We aimed to evaluate the pharmacokinetic and pharmacodynamic effects of a short course (2 weeks) of rifampin on serum etonogestrel (ENG) concentrations and serologic measures of ovarian activity (endogenous estradiol [E2] and progesterone [P4]) among ENG implant users. Study designWe enrolled healthy females using ENG implants for 12 to 36 months. We measured baseline serum ENG concentrations using a validated liquid chromatography mass-spectrometry assay and baseline E2 and P4 concentrations using chemiluminescent immunoassays. After 2 weeks of rifampin 600 mg daily, we repeated ENG, E2, and P4 measurements. We compared pre- and post-rifampin serum measurements using paired Wilcoxon signed-rank tests. ResultsFifteen participants completed all study procedures. Participants’ median age was 28.2 years (range 21.8–34.1), median body-mass index was 25.2 kg/m2 (range 18.9–37.3), and median duration of implant use was 22 months (range 12–32). All participants experienced significant decreases from baseline ENG concentrations (median 164.0 pg/mL [range 94.4–265.0]) to post-rifampin measurements (median 47.8 pg/mL [range 24.7–82.8]) (p < 0.001). Serum E2 concentrations also significantly increased with rifampin exposure (median 73 pg/mL vs 202 pg/mL, p = 0.003); increases in serum P4 concentrations were not statistically significant (p = 0.19). Three participants (20%) experienced increased luteal activity, with one presumptively ovulating post-rifampin (P4 = 15.8 ng/mL). ConclusionsWith only short exposure to a strong CYP3A inducer, ENG implant users experienced clinically significant decreases in serum ENG concentrations that led to changes in biomarkers indicative of waning suppression of ovulation. ImplicationsEven a short, 2-week course of treatment with rifampin places etonogestrel contraceptive implant users at risk for decreased contraceptive efficacy. Clinicians should counsel patients using etonogestrel implants considering any duration of rifampin therapy on the need for backup nonhormonal contraception or the use of an intrauterine device to avoid unintended pregnancies.

Hormonal profile prior to luteolysis modulates the uterine luminal transcriptome in the subsequent cycle in beef cross-bred cows§.

Sex steroid concentrations modulate endometrial function and fertility in cattle. Our objective was to compare the post-estrus luminal transcriptome of cows that were exposed to contrasting concentrations of progesterone (P4) before luteolysis that displayed estrus and ovulated spontaneously. Cross-bred beef cows received either 1) a new CIDR and GnRH (d - 9; high progesterone treatment; HP4; n = 16) or 2) a previously used CIDR, PGF2α, and GnRH (low progesterone treatment; LP4; n = 24). All cows received PGF2α at CIDR removal (d - 2). Ovarian ultrasonography and blood collections were performed on d - 9, d - 2, d - 0.5, d 0 (day of observed estrus), d 4, d 7, and d 14 for measurement of ovarian structures, P4, and estradiol (E2). Luminal epithelial cells were collected using a cytology brush on d 4, d 7, and d 14 for RNAseq. On d - 2, CL area and concentrations of P4 were greater, while on d - 0.5 concentrations of E2 were decreased in HP4. Ovarian structures and hormonal concentrations were similar on d 4, d 7, or d 14 (P > 0.05). There were enriched pathways in HP4 related to activation and signaling of the innate immune system at d 4, downregulation in the network involved in the extracellular matrix remodeling at d 7, and exacerbated inflammatory response as well as differentiation and activation of macrophages at d 14 (Benjamini - Hochberg P-value ≤0.05). In conclusion, manipulation of pre-luteolysis sex steroid concentrations altered the post-estrus luminal transcriptome even though all cows showed estrus and ovulated spontaneously.

Luteal Function, Biometrics, and Echotextural Attributes in Santa Inês Ewes Superovulated with Different Total Doses of Porcine Follicle-Stimulating Hormone

Premature regression of corpora lutea (PRCL) may adversely affect the outcome of hormonal ovarian superstimulation in small ruminants, and the total dose of exogenous gonadotropins used may be one of the causes of this condition. There were two major objectives of the present study: (1) to evaluate the effects of different superovulatory doses of porcine follicle-stimulating hormone (pFSH) on the biometry, blood perfusion (Doppler), and echotextural characteristics of luteal structures; and, (2) to determine the usefulness of biometric, vascular, and echotextural luteal variables, as well as measurements of circulating progesterone (P4) concentrations for early detection of PRCL in superovulated Santa Inês ewes. Twenty-seven Santa Inês ewes received an intravaginal P4-releasing device (CIDR) from Days 0 to 8 (Day 0 = random day of the anovulatory period). An IM injection of d-cloprostenol (37.5 μg) was given at the time of the CIDR insertion and withdrawal. On Day 6, all the ewes received 300 IU of eCG IM and were divided into three treatment groups (each n = 9): G100 (100 mg); G133 (133 mg); and G200 (200 mg of pFSH) administered IM every 12 h in eight injections. Transrectal ovarian ultrasonography and jugular blood sampling for serum P4 measurements were performed on Days 11 to 15. On the day of embryo recovery (Day 15), all the ewes underwent diagnostic videolaparoscopy and were classified, based on their luteal characteristics, into three response groups: nCL (ewes with normal CL only); rCL (ewes with regressing CL only); and ewes with both nCL and rCL following the superovulatory regimen. Our present results indicate that the total pFSH doses of 100 mg and 200 mg result in similar ovulatory responses and luteal function/biometrics, although the percentage of donor ewes with nCL was greater (p < 0.05) for G100 compared with the G200 animals. An application of 133 mg of pFSH was associated with diminished luteogenesis. Lastly, circulating P4 concentrations, ultrasonographic estimates of total luteal area, and CL pixel heterogeneity (standard deviation of numerical pixel values) are promising markers of luteal inadequacy in superovulated ewes.

A drop in serum progesterone from oocyte pick-up +3 days to +5 days in fresh blastocyst transfer, using hCG-trigger and standard luteal support, is associated with lower ongoing pregnancy rates.

A drop in serum progesterone from oocyte pick-up +3 days to +5 days in fresh blastocyst transfer, using hCG-trigger and standard luteal support, is associated with lower ongoing pregnancy rates.

P079Etonogestrel contraceptive implant users can experience breakthrough ovulation with only two weeks of exposure to a strong cyp3a inducer

<h3>Objectives</h3> Rifampin is a strong CYP3A inducer and the gold standard for evaluating CYP3A-mediated drug-drug interactions per the US Food and Drug Administration. We hypothesized that even a short course (two weeks) of exposure would lead to breakthrough ovulation-mediated changes in endogenous estradiol (E2) and progesterone (P4) concentrations among etonogestrel (ENG) implant users. <h3>Methods</h3> We enrolled healthy people using ENG implants for 12–36 months. We measured baseline serum concentrations of E2 and P4 as surrogate biomarkers for ovulation suppression using clinical chemiluminescent immunoassays. After two weeks of rifampin 600 mg daily, we repeated E2 and P4 measurements. We determined breakthrough ovulation based on pre-exposure P4 3.0ng/mL with concurrent rise in post-treatment E2. <h3>Results</h3> Fifteen participants completed all study procedures. Participants' median age was 28.2 (range, 21.8–34.1), median BMI was 25.2 kg/m2 (range, 18.9–37.3), and median duration of implant use was 22 months (range, 12–32). At baseline, median E2 concentrations were 73 pg/mL (range, 32–686) and median P4 concentrations were 0.3 ng/mL (range, 0.2–6.1). After concomitant rifampin, median E2 concentrations were 201 pg/mL (range, 59–951) and median P4 concentrations were 0.4 ng/mL (range, 0.2–15.8). Serum E2 concentrations significantly increased with rifampin exposure (p=0.003, paired Wilcoxon signed rank test), but the increases in serum P4 concentrations were not statistically significant (p=0.185). Three participants (20%) experienced breakthrough ovulation with the following P4 changes: 0.3>5.0, 0.8>7.3, 0.5>15.8 (pre>post; ng/mL). <h3>Conclusions</h3> With only a short exposure to a strong CYP3A inducer, ENG implant users experienced clinically significant changes in hormone biomarkers indicative of waning follicular development and ovulation suppression.