Tanja K. Eggersmann

Background: In anovulatory hormone replacement therapy frozen embryo transfer (HRT-FET) cycles, reproductive success depends entirely on exogenous sex-steroid supplementation. Inadequate progesterone exposure remains a clinically relevant challenge, affecting up to one-third of patients receiving micronized vaginal progesterone (MVP) monotherapy. Combination regimens incorporating oral dydrogesterone (DYD) alongside MVP have been proposed to address this limitation. Critically, the absence of immunoassay cross-reactivity between DYD and progesterone enables simultaneous quantification of both progestins within a single patient – a methodological opportunity not yet exploited in outcome research. Using high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS), we investigated the independent and joint associations of DYD, dihydrodydrogesterone (DHD), progesterone (P), and estradiol (E2) with clinical outcomes in a combination-progestin HRT-FET regimen, establishing a novel pharmacodynamic interaction framework for HRT-FET protocol optimization. Methods: This nested analysis included 111 women undergoing anovulatory HRT-FET within a prospective multicenter cohort (NCT03507673). Patients received oral estradiol (2 mg tid) followed by MVP (400 mg bid) and DYD (10 mg tid) from days 13–15. Serum and plasma samples collected on the day of FET were analyzed using HPLC–MS/MS for DYD and DHD, and immunoassay for P and E2. Clinical pregnancy and live birth were assessed as primary outcomes. Stratified and interaction analyses were performed to explore combined hormone-level effects. Results: Hormone concentrations showed broad interindividual variability with weak inter-analyte correlations (r ≤ 0.33), confirming pharmacodynamic independence of the two progestin pathways. No statistically significant independent association with live birth was observed for any single analyte. However, interaction analyses revealed consistent gradient patterns: live birth rates were highest when both DYD and P concentrations were elevated (67%) and lowest when both were in the lower range (27%). Analogous patterns were observed for DYD–E2 and P–E2 combinations, suggesting additive and substitutive pharmacodynamic interaction effects. Given the hypothesis-generating nature of this study, the sample size is appropriate for the exploratory interaction framework established here. Conclusions: This study introduces simultaneous dual-progestin quantification as a methodological platform for pharmacodynamic interaction research in HRT-FET. Exploratory interaction patterns between both progestins and estradiol suggest clinically relevant additive effects, while patterns between the two progestins are compatible with potential substitutive dynamics that can only be evaluated within combination regimens without analytical cross-reactivity. These findings provide a mechanistic framework and generate hypotheses for adequately powered prospective studies investigating joint hormone exposure and reproductive outcomes. Trial registration: NCT03507673.

STUDY QUESTION: Are previously proposed vaginal microbial community state types (CSTs) valid predictors of IVF success, or do alternative microbial signatures provide stronger associations? SUMMARY ANSWER: Previously proposed CSTs as predictors of implantation, clinical pregnancy, and live birth were not confirmed, while an interaction between Ureaplasma parvum and Lactobacillus iners emerged as a strong negative predictor. WHAT IS KNOWN ALREADY: Infertility affects 17% of the global population. Only one-third of treatment cycles of assisted reproductive technologies result in embryo implantation, and even fewer lead to clinical pregnancy or live births. While early findings have spurred the development of microbiome-based tests for success prediction, evidence on supporting their reliability remains inconclusive. STUDY DESIGN, SIZE, DURATION: This prospective, single-centre study aimed to validate existing, and identify better, microbial predictors of infertility treatment outcomes. A cohort of 266 infertile female patients (age 18–45 years) undergoing a frozen-thawed embryo transfer cycle in an anovulatory regimen (i.e. a cycle with transfer of an embryo following a previous oocyte retrieval, fertilization, and freezing of embryos) was recruited for the study within a timeframe from May 2017 to March 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: The female, infertile patients, aged 18–45 years, were undergoing routine care. Vaginal swabs were taken prior to embryo transfer and subjected to DNA isolation for 16S-based microbiota analysis. Extended demographic and treatment data were recorded. Clinical outcomes were defined as: (i) implantation, confirmed by a positive hCG test, (ii) clinical pregnancy, and (iii) live birth (defined as the birth of a viable infant). Sequencing data were processed in mothur following established pipelines, and microbial composition (taxonomy) as well as microbial diversity (dissimilarity analyses) were determined using the open-source software R. A prediction model for implantation success was built using binary logistic regression based on abundance of putatively predictive microbial taxa. MAIN RESULTS AND THE ROLE OF CHANCE: This study suggests that vaginal microbial CSTs, alpha-diversity, and the ratio of dominant Lactobacillus species do not correlate in statistical terms or in a clinically meaningful manner with implantation and clinical pregnancy (as a surrogate for endometrial receptivity) or with live birth (as a surrogate for ongoing pregnancy viability). However, Ureaplasma parvum and Lactobacillus iners abundances were identified as negative predictors of embryo implantation, clinical pregnancy, and live birth. A subset of women colonized by these taxa experienced drastically reduced embryo implantation and completely failed to achieve clinical pregnancy or give birth to live offspring, suggesting a potential role of these organisms in implantation failure and reproductive outcome, independent of other influencing factors such as age, oestradiol levels, endometrial thickness etc. LARGE SCALE DATA: The raw sequencing data used for this manuscript are publicly available at the European Nucleotide Archive under accession number PRJEB107113. LIMITATIONS, REASONS FOR CAUTION: This study is a single-centre study warranting further validation cohorts. Given the variable nature of the vaginal microbiota, sample sizes need to be enlarged for better refinement of the analyses. Further, the underlying mechanistical basis of our findings is yet elusive and clinical translation has yet to be established. WIDER IMPLICATIONS OF THE FINDINGS: While this novel association warrants confirmation, the results caution against reliance on previously suggested CSTs as predictors, and highlight the need for refined, reproducible microbiome-based diagnostics in reproductive medicine. STUDY FUNDING/COMPETING INTEREST(S): Financial support was received from the University of Lübeck and the German Center of Infection Research. M.L., A.M., I.P., M.D., and J.R. declare no conflicts of interest. S.G. discloses personal fees from Organon outside the submitted work. T.K.E. discloses honoraria from Ferring; travel support from Ferring, Merck, Theramex, and Gedeon-Richter; and receipt of equipment/materials/laboratory analyses (to institution) from Arthrex, Besins, Merck, and Abbott outside the submitted work. N.H. discloses personal fees from Gedeon Richter, Ferring, and Merck. G.G. reports that his institution received grants or contracts from Besins, Merck, Abbott, Ferring, and Theramex. He has received personal consulting fees, support for travel fees and meeting attendance, and honoraria for lectures or educational events from Organon, Ferring, Merck, Gedeon-Richter, Theramex, Abbott, ReproNovo, Igyxos, OxoLife, Philipps, ReprodWissen, PregLem, Guerbet, Roche, IBSA, and Besins. He also received support for travel and meeting attendance from Merck, Organon, Ferring, Theramex, Gedeon-Richter, and Abbott. Additionally, he holds unpaid leadership positions as a member of the ESHRE Working Group on RIF, the ESHRE Working Group on clinical KPI, and the ESHRE guideline development group on ovarian stimulation. A.S.-M. reports consulting fees and speaker’s fees from Merck, Theramex, and Gedeon-Richter as well as travel support from Merck, Theramex, Gedeon-Richter, IBSA, Ferring, and MSD.

STUDY QUESTION: What are the effects of three-times-a-day 10 mg oral dydrogesterone (DYD), initiated in the late follicular phase of natural menstrual cycles to induce endometrial receptivity for frozen–thawed embryo transfer (FET), on progesterone levels, indicative of ovulation on the day of FET, and how are levels of DYD, 20α-dihydrodydrogesterone (DHD), progesterone (P), and estradiol (E2) on the day of FET associated with clinical outcomes? SUMMARY ANSWER: Late follicular phase initiation of oral 30 mg DYD is compatible with progesterone levels indicative of ovulation in 98% of cases, and DYD, DHD, P, and E2 blood levels on the day of FET do not show a consistent relationship with live birth achievement, whether evaluated in isolation or interaction. WHAT IS KNOWN ALREADY: HRT regimens for FET have come under scrutiny due to: (i) the risk of insufficient progesterone exposure with conventional dosing schemes, and (ii) maternal and fetal risks associated with the iatrogenic absence of a corpus luteum. Oral DYD 10 mg three-times-a-day (tid) is considered unlikely to interfere with ovulation or corpus luteum formation and does not exhibit cross-reactivity with progesterone in ELISA. Therefore, it can be used to induce endometrial receptivity (i.e. to schedule the timing of FET in a natural cycle) and provide luteal phase support (LPS) while allowing ovulation to occur independently of the implantation window and enabling the monitoring of endogenous progesterone serum levels. STUDY DESIGN, SIZE, DURATION: Nested within a multi-centric, prospective, clinical cohort study (NCT03507673), 559 normally cycling women from the routine care population who underwent FET in a spontaneous menstrual cycle (12/2021–8/2023) had DYD, DHD, P, and E2 levels on day of FET measured by high-performance liquid chromatography/tandem mass spectroscopy (HPLC/MS/MS) or commercially available electrochemiluminescence immunoassay (ECLIA). PARTICIPANTS/MATERIALS, SETTING, METHODS: Starting from cycle day 10, women underwent endocrine (E2, LH, and P) and sonographic monitoring until the following criteria were ideally met: a leading follicle ≥16 mm, endometrial thickness ≥6 mm, and E2 ≥ 180 pg/ml. At this point, physicians could initiate oral DYD 10 mg (tid) or, if there was no LH rise in serum, postpone initiation within 2 days of monitoring to align with patient and/or centre preferences (e.g. avoiding weekends). FET for Day 2–Day 5 embryos was performed on Day 3–6 of DYD intake. MAIN RESULTS AND THE ROLE OF CHANCE: DYD and DHD plasma levels on Day 3–6 of intake replicate previous findings. In FET with Day 4 or 5 embryos, serum P levels indicated ovulation in 98.1% (>1.5 ng/ml) and 95.4% (>3.0 ng/ml) of cases and levels were higher (ratio of geometric means 2.88, 95% CI 2.48–3.35) than in FET with Day 2 or 3 embryos. Progesterone levels on day of FET were unrelated (ratio of geometric means 1.13, 95% CI 0.95–1.34) to follicular size (≤16 mm vs >16 mm) and only slightly higher (ratio of geometric means 1.36, 95% CI 1.19–1.56) in cases of serum LH level ≥12.6 IU/l versus 25th percentile) hormone levels (DYD RD −5.3%, 95% CI −14.0 to 3.2, P = 0.227; DHD RD −4.0%, 95% CI −14.6 to 6.0, P = 0.428; Progesterone RD 2.9%, 95% CI −7.0 to 12.3, P = 0.597; E2 RD −3.3%, 95% CI −12.2 to 5.3, P = 0.487). These findings were confirmed across the whole range of hormone values by logistic regression, and no interaction effects of the evaluated hormones on live birth rates were found at the unadjusted significance level 0.05. LIMITATIONS, REASONS FOR CAUTION: All outcomes were assessed only in subjects undergoing FET, not on intention-to-treat. Oral DYD 10 mg (tid), similar to other progestins given to induce endometrial receptivity or for LPS, will likely still interfere with the LH surge, ovulation, and luteal phase characteristics, warranting further investigations using a true natural cycle (NC) as the reference. WIDER IMPLICATIONS OF THE FINDINGS: We propose the term ‘Programmed-Ovulatory (PO-) FET’ for this protocol. The PO-FET protocol enables scheduling the window of implantation, allows ovulation and corpus luteum formation, provides double gestagenic support, and may eliminate the need for control measurements of sex steroids on the day of FET. It allows monitoring of corpus luteum activity during the luteal phase and early pregnancy, is injection-free, and has low drug costs. This protocol needs to be tested in RCTs against other ovulatory FET protocol options, such as the ‘NC FET’ or ‘managed NC FET’. STUDY FUNDING/COMPETING INTEREST(S): The trial was funded through institutional resources of the University Hospital of Schleswig-Holstein, Campus Lübeck. Expenses related to plasma and serum sample handling, storage, shipment, and the HPLC/MS/MS and ELISA analyses of DYD, DHD, estradiol, and progesterone were financially supported by Abbott Products Operations AG (Allschwil, Switzerland). The funding was provided in the form of a research grant to the Department of Gynecological Endocrinology and Reproductive Medicine, University Hospital of Schleswig-Holstein, Campus Lübeck (no grant number applicable). Abbott Products Operations AG had no role in the study design, conduct, data collection, statistical analysis, data interpretation, manuscript preparation, or the decision to submit the article for publication. T.K.E. discloses honoraria from Ferring; travel support from Merck, Ferring, Theramex, and Gedeon-Richter; and receipt of equipment/materials (to institution) from Arthrex and Besins Healthcare, outside the submitted work. N.H. has received travel support from Gedeon-Richter, Ferring, and Merck, outside the submitted work. A.R.H. has received honoraria from Organon and travel support from Merck Serono, Gedeon Richter, and Theramex,...

Objectives Progesterone, a sex steroid, is measured in serum by immunoassay in a variety of clinical contexts. One potential limitation of steroid hormone immunoassays is interference caused by compounds with structural similarity to the target steroid of the assay. Dydrogesterone (DYD), an orally active stereoisomer of progesterone, is used for various indications in women’s health. Herein, we report a systematic in vitro investigation of potential interference of DYD and its active metabolite 20α-dihydrodydrogesterone (DHD) in seven widely used, commercially available progesterone assays. Methods Routine human plasma samples were anonymized and pooled to create three graded concentration levels of progesterone (P4 high, P4 medium, P4 low). Each pooled P4 plasma sample (6–7 mL) was spiked at high, medium, and “none” concentration with DYD/DHD and was divided into 0.5 mL aliquots. The blinded aliquots were analyzed by seven different laboratories with their routine progesterone assay (six different immunoassays and one liquid chromatography–tandem mass spectrometry assay, respectively) within the Dutch working group on endocrine laboratory diagnostics of the Dutch Foundation for Quality Assessments in Medical Laboratories. Results The sample recovery rate (P4 result obtained for sample spiked with DYD/DHD, divided by the result obtained for the corresponding sample with no DYD/DHD × 100) was within a ±10% window for the medium and high P4 concentrations, but more variable for the low P4 samples. The latter is, however, attributable to high inter- and intra-method variability at low P4 concentrations. Conclusions This study does not indicate any relevant interference of DYD/DHD within routinely used progesterone assays.

Introduction: This project aimed to identify the frequency of a switch of hormone receptor (HR) and/or HER2 status after neoadjuvant chemotherapy (NAC) for early breast cancer. Methods: Tumor samples from patients without pathological complete response (non-pCR) were evaluated. Pathological complete response (pCR) was defined as no invasive tumor in breast and lymph nodes (ypT0/is ypN0). HR and HER2 status determined before NAC was compared with the corresponding receptor status determined in the surgical specimen after NAC. Results: 245 consecutive patients with primary invasive breast cancer, treated with NAC with/without targeted therapy between January 1, 2016 and December 31, 2019, at the LMU Breast Center, Munich, Germany, were identified. In 128 patients (52%), surgery revealed non-pCR after completed NAC. In 35 cases (27%), a switch of either HR and/or HER2 status between the initial biopsy and the surgical specimen was detected. Twenty cases had a switch in HR status, while 15 cases had a switch in HER2 status. Conclusion: In a substantial number (27%) of non-pCR cases, a switch in biomarker status after completed neoadjuvant treatment was detected. These results are consistent with prior evidence. Yet, routine reevaluation of HR and HER2 status is not recommended in guidelines so far. Future research needs to address the impact of HR and HER2 status switch on therapy adaptation and on subsequent patient outcome. Particularly, in view of the recent therapy advances, it will be critical to evaluate whether individualization of treatment concepts based on the biology of the non-pCR specimens is preferable to the initial therapy concept based on the pathology at primary diagnosis.

Molecular factors that drive metastasis in premenopausal patients with hormone receptor positive (HR+), human epidermal growth factor receptor 2 negative (HER2−), early breast cancer (EBC) are largely unknown. To identify markers/signatures contributing to metastasis, we analyzed molecular changes in tumors from premenopausal patients who developed metastasis (M1) and who did not (M0). Ninety-seven premenopausal patients with HR+/HER2− EBC were included (M1, n = 48, median distant metastasis-free survival (DMFS): 54 (7–184) months; M0, n = 49, median follow-up: 149 (121–191) months). Gene expression profiling on tumor RNA (Breast Cancer 360^TM panel, Nanostring) was performed, followed by comprehensive bioinformatic and statistical analyses. Significantly enhanced ROR (risk of recurrence) scores and reduced signature scores of PGR (progesterone receptor), claudin-low, and mammary stemness were determined in M1. These differences were significantly associated with shorter DMFS in univariate survival analyses. Gene set enrichment analysis showed an enriched mTORC1 pathway in M1. Moreover, a metastasis signature of 19 differentially expressed genes (DEGs) that were DMFS-related was defined. Multivariate analysis including the four signatures, 19 DEGs, pN, and pT status, identified LRP2, IBSP, and SCUBE2 as independent prognostic factors. We identified prognostic gene signatures and single-gene markers for distant metastasis in premenopausal HR+/HER2− EBC potentially applicable in future clinical practice.

Human adipose-derived stem/stromal cells (ASCs) are increasingly used as auto-transplants in regenerative medicine to restore tissue defects or induce wound healing, especially in cancer patients. The impact of ASCs on squamous cell carcinoma of the upper aerodigestive tract (UAT) including head and neck and esophageal squamous cell carcinoma (HNSCC and ESCC) is not yet fully understood. ASCs were cultured from subcutaneous, abdominal lipoaspirates of five patients, who received auto-transplants to the head and neck. Supernatants were tested for paracrine effects in functional in vitro assays of proliferation of HNSCC tumor cell line FaDu and ESCC cell line Kyse30, and their cell migration/invasion capacities in Boyden chambers, in addition to endothelial tube formation assay using human umbilical vein endothelial cells (HUVECs). All ASC-derived supernatants enhanced proliferation of FaDu cells, invasive migration, and tube formation by HUVECs, compared to controls. Of five patients’ lipoaspirates, ASC-derived supernatants of four patients increased proliferation and invasive migration in Kyse30 cells. The data suggests that ASCs can promote tumor cell proliferation, invasiveness, and neo-angiogenesis in these tumor cell lines of the UAT and HUVEC in a paracrine manner. Although clinical studies on the subject of oncological safety are still needed, these findings emphasize the importance of complete tumor removal before ASCs are used in the head and neck.

The majority of patients with metastatic breast cancer (MBC) have hormone receptor-positive HER2-negative disease. For this subgroup, endocrine therapy is the key therapeutic option. Recently, therapeutic options have been expanded by introduction of the inhibitors of cyclin-dependent kinases 4/6 (CDK4/6i). Three compounds, palbociclib, ribociclib, and abemaciclib, have already been approved by the FDA for use together with endocrine therapy such as aromatase inhibitors (AIs) or fulvestrant; abemaciclib is also approved as a single agent. In the first-line setting, all three agents—together with an AI—substantially prolonged progression-free survival with a consistent hazard ratio of around 0.5 in all phase III trials. The data for second-line settings and beyond is also quite consistent, with again a substantial prolongation of progression-free survival demonstrated for fulvestrant together with palbociclib, ribociclib, or abemaciclib. Treatment with CDK4/6i is well tolerated and side effects are manageable. With palbociclib and ribociclib, hematological toxicities are most frequent. Abemaciclib has a lower incidence of neutropenia and a much greater incidence of all grades of diarrhea compared with other CDK4/6i, making diarrhea the key toxicity for abemaciclib. Patient quality of life is maintained under therapy and, particularly in later line settings, deterioration of quality of life is slowed down and symptoms such as pain are better controlled by CDK4/6i. Their consistent and clinically relevant efficacy makes these drugs an important improvement in our armamentarium against MBC and, potentially, ideal candidates in early breast cancer (EBC). This review summarizes the available clinical data for CDK4/6i and current research activities, particularly in EBC.

In an outpatient setting, some challenges of cancer treatment include continuous patient–physician communication, lack of adherence, potential side effects and their impact on quality of life and other patient-reported outcomes. These challenges in the support of disease management can be overcome by the introduction of eHealth applications. Though the market of eHealth applications is fast growing, many applications lack evidence regarding their effectiveness, safety and utility. Only few prospective randomized trials have been conducted, so far. Results of these studies univocally show a gain in health-related quality of life, in the examined eHealth applications. It remains unclear if procedural and cost efficacy are affected by eHealth applications. The upcoming PreCycle study will be the largest randomized eHealth study in oncology.