Mitochondrial DNA variants segregate during human preimplantation development into genetically different cell lineages that are maintained postnatally.

Humans present remarkable diversity in their mitochondrial DNA (mtDNA) in terms of variants across individuals as well as across tissues and even cells within one person. We have investigated the timing of the first appearance of this variant-driven mosaicism. For this, we deep-sequenced the mtDNA of 254 oocytes from 85 donors, 158 single blastomeres of 25 day-3 embryos, 17 inner cell mass and trophectoderm samples of 7 day-5 blastocysts, 142 bulk DNA and 68 single cells of different adult tissues. We found that day-3 embryos present blastomeres that carry variants only detected in that cell, showing that mtDNA mosaicism arises very early in human development. We classified the mtDNA variants based on their recurrence or uniqueness across different samples. Recurring variants had higher heteroplasmic loads and more frequently resulted in synonymous changes or were located in non-coding regions than variants unique to one oocyte or single embryonic cell. These differences were maintained through development, suggesting that the mtDNA mosaicism arising in the embryo is maintained into adulthood. We observed a decline in potentially pathogenic variants between day 3 and day 5 of development, suggesting early selection. We propose a model in which closely clustered mitochondria carrying specific mtDNA variants in the ooplasm are asymmetrically distributed throughout the cell divisions of the preimplantation embryo, resulting in the earliest form of mtDNA mosaicism in human development.

High-impact journal publishing: the devil is in the detail!

Research in medicine is an indispensable tool to advance knowledge and improve patient care. This may be particularly true in the field of human reproduction as it is a relatively new field and treatment options are rapidly evolving. This is of particular importance in an emerging field like "human reproduction", where treatment options evolve fast.The cornerstone of evidence-based knowledge, leading to evidence-based treatment decisions, is randomized controlled trials as they explore the benefits of new treatment approaches. The study design and performance are crucial and, if they are carried out correctly, solid conclusions can be drawn and be implemented in daily clinical routines. The dissemination of new findings throughout the scientific community occurs in the form of publications in scientific journals, and the importance of the journal is reflected in part by the impact factor. The peer review process before publication is fundamental in preventing flaws in the study design. Thus, readers of journals with a high impact factor usually rely on a thorough peer review process and therefore might not question the published data. However, even papers published in high-impact journals might not be free of flaws, so the aim of this paper is to encourage readers to be aware of this fact and critically read scientific papers as 'the devil lies in the details'.

Circadian serum progesterone variations on the day of frozen embryo transfer in artificially prepared cycles.

RESEARCH QUESTION: What is the intra-day variation of serum progesterone related to vaginal progesterone administration on the day of frozen embryo transfer (FET) in an artificial cycle? DESIGN: A prospective cohort study was conducted including 22 patients undergoing a single blastocyst artificial cycle (AC)-FET from August to December 2022. Endometrial preparation was achieved by administering oestradiol valerate (2 mg three times daily) and consecutively micronized vaginal progesterone (MVP; 400 mg twice daily). A blastocyst FET was performed on the 6th day of MVP administration. Serum progesterone concentrations were measured on the day of transfer at 08:00, 12:00, 16:00 and 20:00 hours. The first and last blood samples were collected just before MVP was administered. RESULTS: The mean age and body mass index of the study population were 33.95 ± 3.98 years and 23.10 ± 1.95 kg/m2. The mean P-values at 08:00, 12:00, 16:00 and 20:00 hours were 11.72 ± 4.99, 13.59 ± 6.33, 10.23 ± 3.81 and 9.28 ± 3.09 ng/ml, respectively. A significant decline, of 2.41 ng/ml (95% confidence interval 0.81-4.00), was found between the first and last progesterone measurements. CONCLUSION: A statistically significant intra-day variation of serum progesterone concentrations on the day of FET in artificially prepared cycles was observed. This highlights the importance of a standardized procedure for the timing of progesterone measurement on the day of AC-FET. Of note, the study results are applicable only to women using MVP for luteal phase support; therefore it is necessary to confirm its validity in comparison with the different existing administration routes of progesterone.

Early versus late follicular phase ovarian stimulation: a randomized controlled trial.

RESEARCH QUESTION: Is late follicular phase stimulation as efficient as early follicular phase stimulation in a gonadotrophin-releasing hormone (GnRH) antagonist protocol in oocyte donors in terms of the number of oocytes. DESIGN: In this open label, phase 3, non-inferiority, randomized controlled trial using a two-arm design with a 1:1 allocation ratio, 84 oocyte donors were allocated to the early follicular start group (control group, n = 41) or the late follicular start group (study group, n = 43). In the control group, women followed a fixed GnRH antagonist protocol with recombinant FSH (r-FSH) 225 IU. In the study group, r-FSH 225 IU was initiated in the late follicular phase. The primary outcome was the number of oocytes. The secondary outcomes were the number of mature oocytes, consumption of gonadotrophins and GnRH antagonist, and cost of medication. RESULTS: The number of oocytes did not differ between the control group and the study group (intent-to-treat analysis 15.5 ± 11.0 versus 14.0 ± 10.7, P = 0.52; per-protocol analysis 18.2 ± 9.7 versus 18.8 ± 7.8, P = 0.62). In addition, the number of mature oocytes did not differ between the groups (14.1 ± 8.1 versus 12.7 ± 8.5, P = 0.48). The duration of stimulation was shorter in the control group (10.0 ± 1.4 versus 10.9 ± 1.5 days, P = 0.01). The total amount of r-FSH used was lower in the control group (2240.7 ± 313.9 IU versus 2453.9 ± 330.1 IU, P = 0.008). A GnRH antagonist was used for approximately 6 days in the control group, while a GnRH antagonist was only prescribed for one woman in the study group (6.0 ± 1.4 days versus 0.13±0.7 days, P < 0.001). There was a significant difference in the cost of medication per cycle between the groups (1147.9 ± 182.8€ in control group versus 979.9 ± 129.0€ in study group, P < 0.001). CONCLUSIONS: Late follicular phase stimulation is as efficient as early follicular phase stimulation in terms of the number of oocytes.

IVF with reception of oocytes from partner in lesbian couples: a systematic review and SWOT analysis.

The growing utilization of assisted reproductive technology (ART) by the LGBTQ+ community, especially among lesbian couples, challenges societal norms and promotes inclusivity. The reception of oocytes from partner (ROPA) technique enables both female partners to have a biological connection to their child. A systematic review was conducted of the literature on ROPA IVF to provide the latest data and a SWOT analysis was subsequently performed to understand the strengths, weaknesses, opportunities and threats associated with ROPA IVF. Publications from 2000 to 2023 with relevant keywords were reviewed and 16 records were included. Five studies provided clinical information on couples who used ROPA IVF. ROPA IVF provides a unique opportunity for a biological connection between the child and both female partners and addresses concerns related to oocyte donation and anonymity. Weaknesses include limited cost-effectiveness data and unresolved practical implications. Opportunities lie in involving both partners in parenthood, advancing ART success rates and mitigating risks. Threats encompass increased pregnancy complications, ethical concerns, insufficient safety data, legal or cultural barriers, and emotional stress. In conclusion, ROPA IVF offers a promising solution for lesbian couples seeking to create a family in which both partners want to establish a biological connection with their child.

Association of severity of menstrual dysfunction with cardiometabolic risk markers among women with polycystic ovary syndrome.

INTRODUCTION: Polycystic ovary syndrome (PCOS) is associated with a wide range of unfavorable cardiometabolic risk factors, including obesity, hypertension, insulin resistance, impaired glucose metabolism, dyslipidemia, and metabolic syndrome. Compared with women with regular menstrual cycles, women with a history of irregular menstrual periods have an increased unfavorable cardiometabolic risk. Recently, the association between the severity of oligomenorrhea and hyperinsulinemia and insulin resistance has been demonstrated. However, evidence linking the severity of menstrual cyclicity with cardiometabolic risk in PCOS women is scarce. MATERIAL AND METHODS: This work was a prospective cross-sectional study. A total of 154 women diagnosed with PCOS by the Rotterdam criteria were recruited from July 2021 to September 2022. PCOS women with eumenorrheic (eumeno group), oligomenorrhea (oligo group), and amenorrhea (ameno group) underwent history and physical examination, gonadal steroid hormone measurement, lipid profile, oral glucose tolerance test, and homeostasis model assessment of insulin resistance. RESULTS: A trend toward an increase in unfavorable cardiometabolic risk markers including obesity, hypertension, prevalence of insulin resistance, prediabetes, dyslipidemia, and metabolic syndrome was observed in the ameno group (n = 57) as compared with the eumeno (n = 24) or oligo group (n = 73). A higher prevalence of insulin resistance (odds ratio [OR]: 3.02; 95% confidence interval [CI]: 1.03-8.81) and prediabetes (OR: 3.94; 95% CI: 1.01-15.40) was observed in the ameno group than in the eumeno group, and a higher proportion of dyslipidemia (OR: 2.44; 95% CI: 1.16-5.15) was observed in the ameno group than in the oligo group in the binary logistic regression analysis after adjusting for confounding factors. CONCLUSIONS: PCOS women with amenorrhea show a higher prevalence of insulin resistance, prediabetes, and dyslipidemia compared with those with oligomenorrhea or eumenorrhea. The severity of menstrual dysfunction could be used as a readily obtainable marker for the identification of PCOS women at greatest risk of cardiometabolic diseases.

A heatmap for expected cumulative live birth rate in preimplantation genetic testing for monogenic disorders and chromosomal structural rearrangements.

PURPOSE: Our objective is to predict the cumulative live birth rate (CLBR) and identify the specific subset within the population undergoing preimplantation genetic testing for monogenic disorders (PGT-M) and chromosomal structural rearrangements (PGT-SR) which is likely to exhibit a diminished expected CLBR based on various patient demographics. METHODS: We performed a single-centre retrospective cohort study including 1522 women undergoing 3130 PGT cycles at a referral centre for PGT. A logistic regression analysis was performed to predict the CLBR per ovarian stimulation in women undergoing PGT-M by polymerase chain reaction (PCR) or single-nucleotide polymorphism (SNP) array, and in women undergoing PGT-SR by SNP array, array comparative genomic hybridization (CGH) or next-generation sequencing (NGS). RESULTS: The mean age of women was 32.6 years, with a mean AMH of 2.75 µg/L. Female age and AMH significantly affected the expected CLBR irrespective of the inheritance mode or PGT technology. An expected CLBR < 10% was reached above the age of 42 years and AMH ≤ 1.25 µg/L. We found no significant difference in outcome per ovarian stimulation between the different PGT technologies, i.e. PCR, SNP array, array CGH and NGS. Whereas per embryo transfer, we noticed a significantly higher probability of live birth when SNP array, array CGH and NGS were used as compared to PCR. CONCLUSION: In a PGT-setting, couples with an unfavourable female age and AMH should be informed of the prognosis to allow other reproductive choices. The heatmap produced in this study can be used as a visual tool for PGT couples.

Clinical pregnancy rate for frozen embryo transfer with HRT: a randomized controlled pilot study comparing 1 week versus 2 weeks of oestradiol priming.

RESEARCH QUESTION: Does a frozen-embryo transfer in an artificially-prepared endometrium (FET-HRT) cycle yield similar clinical pregnancy rate with 7 days of oestrogen priming compared to 14 days? DESIGN: This is a single-centre, randomized, controlled, open-label pilot study. All FET-HRT cycles were performed in a tertiary centre between October 2018 and January 2021. Overall, 160 patients were randomized, with a 1:1 allocation, into two groups of 80 patients each: group A (7 days of E2 prior to P4 supplementation) and group B (14 days of E2 prior to P4 supplementation). Both groups received single blastocyst stage embryos on the 6th day of vaginal P4 administration. The primary outcome was the feasibility of such strategy assessed as clinical pregnancy rate, secondary outcomes were biochemical pregnancy rate, miscarriage rate, live birth rate and serum hormone levels on the day of FET. Chemical pregnancy was assessed by an hCG blood test 12 days after FET and clinical pregnancy was confirmed by transvaginal ultrasound at 7 weeks. RESULTS: The analysis included 160 patients who were randomly assigned to either group A or group B on the seventh day of their FET-HRT cycle if the measured endometrial thickness was above 6.5 mm. Following screening failures and of drop-outs, 144 patients were finally included both in group A (75 patients) or group B (69 patients). Demographic characteristics for both groups were comparable. The biochemical pregnancy rate was 42.5% and 48.8% for group A and group B, respectively (p 0.526). Regarding the clinical pregnancy rate at 7 weeks, no statistical difference was observed (36.3% vs 46.3% for group A and group B, respectively, p = 0.261). The secondary outcomes of the study (biochemical pregnancy, miscarriage, and live birth rate) were comparable between the two groups for IIT analysis, as well as the P4 values on the day of FET. CONCLUSIONS: In a frozen embryo transfer cycle, performed with artificial preparation of the endometrium, 7 versus 14 days of oestrogen priming are comparable, in terms of clinical pregnancy rate; the advantages of a seven-day protocol include the shorter time to pregnancy, reduced exposure to oestrogens, and more flexibility of scheduling and programming, and less probability to recruit a follicle and have a spontaneous LH surge. It is important to keep in mind that this study was designed as a pilot trial with a limited study population as such it was underpowered to determine the superiority of an intervention over another; larger-scale RCTs are warranted to confirm our preliminary results. TRIAL REGISTRATION: Clinical trial number: NCT03930706.

Avoiding weekend frozen embryo transfer in modified natural cycles: is it possible?

In this era of the freeze-all strategy, the prevalence of frozen embryo transfer (FET) cycles is increasing rapidly. Although still quite often used, the hormone replacement therapy cycle to prepare a FET should now belong to the past, unless strictly necessary. This raises questions about possible flexible protocols for the preparation of an FET cycle in a (modified) natural cycle. In this viewpoint, an overview of the different options is discussed, stressing the importance of the corpus luteum.

The impact of war on reproductive medicine in Ukraine.

Reproductive medicine plays a vital role in ensuring the health and well-being of individuals and families. However, the ongoing assault of Russia on Ukraine has disrupted the provision of healthcare services in Ukraine, including of reproductive medicine. The impact of the war on the state of reproductive medicine can provide valuable insights into the challenges faced by healthcare providers and policy makers in war-affected areas. This article explores the situation of reproductive medicine in Ukraine after the Russian attack on Ukraine and the full-scale war on 24 February 2022, including changes in the availability and quality of services and the challenges faced by healthcare providers and patients. IVF clinics face significant challenges in safeguarding the fate of cryopreserved embryos and reproductive cells. To address these challenges, clinics need to take measures to ensure their facilities' safety and security, maintain continuity of operations, and navigate ethical and legal dilemmas related to patients' reproductive material. By examining the available data and insights from healthcare providers and patients, this article will contribute to the broader understanding of the impact of war on reproductive health and the importance of ensuring access to essential healthcare services in war-affected areas.

Should we stop screening for chronic endometritis?

Chronic endometritis is a poorly understood infectious or inflammatory process, potentially disrupting the correct implantation of a human embryo (Puente et al., 2020). The exact prevalence is a subject of discussion and ranges across the available literature from 2% to almost 60%, with a higher suspicion of the condition being present in women with recurrent early pregnancy loss and recurrent implantation failure (Puente et al., 2020). The impact of chronic endometritis on reproductive outcomes following IVF remains questionable given the lack of proper data convincingly showing an improvement after diagnosis and treatment. This article aims to provide the reader with a critical appraisal of current diagnostic methods, treatments and patient populations to be tested for chronic endometritis.

The time has come for harmonized international ART registration.

For more than two decades, the European IVF-Monitoring Consortium has collected data on IVF in Europe with the aim of monitoring the quality and safety of assisted reproductive technology (ART) treatments, to ensure the highest performance with the lowest risk for patients and their offspring. Likewise, the Society for Assisted Reproductive Technology in the USA and the Australia/New Zealand Assisted Reproduction Database collect, process and publish data in their regions. The better the legal framework for ART surveillance, the more complete and reliable are the datasets. Worldwide, the landscape of ART regulation is fragmented, and until there is a legal obligation to report ART data in all countries, with an appropriate quality control of the data collected, the reported outcomes should be interpreted with caution. Once uniform and harmonized data are achieved, consensus reports based on collective findings can begin to address key topics such as cycle segmentation and complications. Improved registration systems and datasets allowing optimized surveillance should be developed in collaboration with patient representatives to consider patients' needs, especially aiming to provide higher transparency around ART services. Support from national and international reproductive medicine societies will also be essential to the future evolution of ART registries.

Individualized luteal phase support using additional oral dydrogesterone in artificially prepared frozen embryo transfer cycles: is it beneficial?

RESEARCH QUESTION: Does additional supplementation with oral dydrogesterone improve reproductive outcomes in patients with low serum progesterone concentrations on the day of frozen embryo transfer (FET) after artificial (HRT) endometrial preparation? DESIGN: Retrospective, single-centre cohort study including 694 unique patients performing single blastocyst transfer in an HRT cycle. For luteal phase support, intravaginal micronized vaginal progesterone (MVP, 400 mg twice daily) was administered. Serum progesterone concentrations were assessed prior to FET and outco-mes were compared among patients with normal serum progesterone (≥8.8 ng/ml) continuing the routine protocol and patients with low serum progesterone (<8.8 ng/ml) who received additional oral dydrogesterone supplementation (10 mg three times daily) from the day after FET onwards. Primary outcome was live birth rate (LBR), with a multivariate regression model correcting for relevant confounders. RESULTS: Normal serum progesterone concentrations were observed in 547/694 (78.8%) of patients who continued only MVP as planned, whereas low (<8.8 ng/ml) serum progesterone concentrations were detected in 147/694 (21.2%) patients who received additional oral dydrogesterone supplementation on top of MVP from the day after FET onwards. LBR was comparable between both groups: 37.8% for MVP-only versus 38.8% for MVP+OD (P = 0.84). The multivariate logistic regression model indicated that LBR was not significantly associated with the investigated approaches (adjusted odds ratio 1.01, 95% confidence interval 0.69-1.47, P = 0.97). CONCLUSIONS: The current findings suggest that additional oral dydrogesterone supplementation in patients with low serum progesterone concentrations at the moment of transfer could have the potential to rescue reproductive outcomes in HRT-FET cycles. This field of research, however, remains hampered by the absence of randomized controlled trials.

Multicentre study on rates and reasons for treatment discontinuation in patients with remaining cryopreserved embryos.

RESEARCH QUESTION: What is the discontinuation rate among patients with remaining cryopreserved embryos in Belgium and what are the reasons for discontinuation? DESIGN: Multicentre, cross-sectional study across 11 Belgian fertility clinics. Patients were eligible (n = 1917) if they had previously undergone an unsuccessful fresh embryo transfer (fresh group) or frozen embryo transfer (FET) (in-between group) and did not start a subsequent FET cycle within 1 year despite having remaining cryopreserved embryos. The denominator was all patients with embryos cryopreserved during the same period (2012-2017) (n = 21,329). Data were collected through an online anonymous questionnaire. RESULTS: The discontinuation rate for patients with remaining cryopreserved embryos was 9% (1917/21329). For the final analysis, 304 completed questionnaires were included. The most important reasons for discontinuing FET cycles were psychological (50%) and physical (43%) burden, effect on work (29%), woman's age (25%) and effect on the relationship (25%). In 69% of cases, the patient themselves made the decision to delay FET treatment. In 16% of respondents, the decision to delay FET was determined by external factors: treating physician (9%), social environment (4%), close family (3%) and society (3%). Suggested improvements were psychological support before (41%), during (51%) and after (51%) treatment, as well as lifestyle counselling (44%) and receiving digital information (43%). CONCLUSIONS: The discontinuation rate is remarkably high in patients with remaining cryopreserved embryos who have a good prognosis. Respondents stressed the need to improve the integration of psychological and patient-tailored care into daily assisted reproductive technology practice.

Association between sequence variants in the FSHR gene and reproductive outcomes following IVF in predicted normoresponders.

RESEARCH QUESTION: Is there an association between FSHR sequence variants and reproductive outcomes following IVF in predicted normoresponders? DESIGN: Multicentre prospective cohort study conducted from November 2016 to June 2019 in Vietnam, Belgium and Spain including patients aged <38 years, and undergoing IVF with a predicted normal response with fixed-dose 150 IU rFSH in an antagonist protocol. Genotyping was performed for three FSHR (c.919A>G, c.2039A>G, c.-29G>A) and one FSHB sequence variants (c.-211G>T). Clinical pregnancy rate (CPR), live birth rate (LBR) and miscarriage rate in the first embryo transfer and cumulative live birth rate (CLBR) were compared between the different genotypes. RESULTS: A total of 351 patients underwent at least one embryo transfer. Genetic model analysis that adjusted for patient age, body mass index, ethnicity, type of embryo transfer, embryo stage and number of top-quality embryos transferred revealed a higher CPR for homozygous patients for the variant allele G of c.919A>G when compared to patients with genotype AA (60.3% versus 46.3%, adjusted odds ratio [ORadj] 1.96, 95% confidence interval [CI] 1.09-3.53). Also, c.919A>G genotypes AG and GG presented a higher CPR and LBR when compared with genotype AA (59.1% versus 46.3%, ORadj 1.80, 95% CI 1.08-3.00, and 51.3% versus 39.0%, ORadj 1.69, 95% CI 1.01-2.80, respectively). Cox regression models revealed a statistically significantly lower CLBR for c.2039A>G genotype GG in the codominant model (hazard ratio [HR] 0.66, 95% CI 0.43-0.99). CONCLUSION: These results demonstrate a previously unreported association between variant c.919A>G genotype GG and higher CPR and LBR in infertile patients and reinforce a potential role for genetic background in predicting the reproductive prognosis following IVF.

Double stimulation for the management of poor-prognosis patients: where are we going?

PURPOSE OF REVIEW: The technical improvements in IVF allowed the implementation of nonconventional ovarian stimulation protocols for some specific patients. Where time is crucial, such as with oncologic patients, poor-prognosis patients, patients with low ovarian reserve, and those with advanced maternal age, access to IVF treatment is even more critical. Some of these protocols might start in the late follicular phase, luteal phase, or involve both stimulations within the same ovarian cycle. RECENT FINDINGS: Until now, published evidence showed that oocytes retrieved from unconventional protocol seem to be developmentally, genetically, and reproductively competent. Second stimulation in the same ovarian cycle after the conventional approach may represent a sound alternative to oocyte accumulation. This can be proposed in progress after careful counselling focused on the patients' chances of finding at least one euploid embryo on account of their age and of the number of blastocysts obtained after the conventional approach. SUMMARY: The adoption of these new strategies, known as double stimulation protocol, can be conceived as a real full-personalization of ovarian stimulation. Multicentre prospective RCTs are urgently needed to evaluate the efficacy, efficiency, and costs of double stimulation versus two consecutive conventional approaches with standard or mild stimulation and in a different IVF setting.

Progesterone levels during ovarian stimulation in segmented ART cycles inversely correlate with normalized birthweight of neonates conceived through subsequent frozen-thawed embryo transfer.

PURPOSE: To evaluate the association between serum progesterone (P) at the day of ovulation trigger and neonatal birthweight in singletons born after frozen-thawed embryo transfer in segmented ART cycles. METHODS: A retrospective multicenter cohort study involving data from patients who achieved uncomplicated pregnancy and term delivery of ART-conceived singleton babies following a segmented GnRH antagonist cycle. The main outcome was birthweight's z-score of the neonate. Univariate and multivariate linear logistic regression analyses were made to investigate the relation of z-score with variables inherent to the patient and to the ovarian stimulation. The variable P per oocyte was created by dividing the value of progesterone at ovulation trigger by the number of oocytes retrieved at oocyte retrieval. RESULTS: A total of 368 patients were included in the analysis. At univariate linear regression, the birthweight z-score of the neonate appeared to be inversely related to both P levels at the ovulation trigger (- 0.101, p = 0.015) and P levels per oocyte at trigger (- 1.417, p = 0.001), while it was directly related to the height of the mother (0.026, p = 0.002) and to the number of previous live births (0.291, p = 0.016). In multivariate analysis, both serum P (- 0.1; p = 0.015) and P per oocyte (- 1.347, p = 0.002) maintained the significant inverse association with birthweight z-score after adjusting for height and parity. CONCLUSIONS: Serum progesterone level on the day of ovulation trigger inversely correlates with normalized birthweight of neonates in segmented GnRH antagonist ART cycles.

Comparing Vaginal and Endometrial Microbiota Using Culturomics: Proof of Concept.

It is generally accepted that microorganisms can colonize a non-pathological endometrium. However, in a clinical setting, endometrial samples are always collected by passing through the vaginal-cervical route. As such, the vaginal and cervical microbiomes can easily cross-contaminate endometrial samples, resulting in a biased representation of the endometrial microbiome. This makes it difficult to demonstrate that the endometrial microbiome is not merely a reflection of contamination originating from sampling. Therefore, we investigated to what extent the endometrial microbiome corresponds to that of the vagina, applying culturomics on paired vaginal and endometrial samples. Culturomics could give novel insights into the microbiome of the female genital tract, as it overcomes sequencing-related bias. Ten subfertile women undergoing diagnostic hysteroscopy and endometrial biopsy were included. An additional vaginal swab was taken from each participant right before hysteroscopy. Both endometrial biopsies and vaginal swabs were analyzed using our previously described WASPLab-assisted culturomics protocol. In total, 101 bacterial and two fungal species were identified among these 10 patients. Fifty-six species were found in endometrial biopsies and 90 were found in vaginal swabs. On average, 28 % of species were found in both the endometrial biopsy and vaginal swab of a given patient. Of the 56 species found in the endometrial biopsies, 13 were not found in the vaginal swabs. Of the 90 species found in vaginal swabs, 47 were not found in the endometrium. Our culturomics-based approach sheds a different light on the current understanding of the endometrial microbiome. The data suggest the potential existence of a unique endometrial microbiome that is not merely a presentation of cross-contamination derived from sampling. However, we cannot exclude cross-contamination completely. In addition, we observe that the microbiome of the vagina is richer in species than that of the endometrium, which contradicts the current sequence-based literature.

A randomized, controlled, first-in-patient trial of choriogonadotropin beta added to follitropin delta in women undergoing ovarian stimulation in a long GnRH agonist protocol.

STUDY QUESTION: Does addition of choriogonadotropin beta (recombinant CG beta) to follitropin delta increase the number of good-quality blastocysts following ovarian stimulation in a long GnRH agonist protocol? SUMMARY ANSWER: At the doses investigated, the addition of CG beta reduced the number of intermediate follicles and related down-stream parameters including the number of oocytes and blastocysts. WHAT IS KNOWN ALREADY: CG beta is a novel recombinant hCG (rhCG) molecule expressed by a human cell line (PER.C6®) and has a different glycosylation profile compared to urinary hCG or rhCG derived from a Chinese Hamster Ovary (CHO) cell line. In the first-in-human trial, the CG beta pharmacokinetics were similar between men and women. In women, the AUC and the peak serum concentration (Cmax) increased approximately dose proportionally following single and multiple daily doses. In men, a single dose of CG beta provided higher exposure with a longer half-life and proportionately higher testosterone production than CHO cell-derived rhCG. STUDY DESIGN, SIZE, DURATION: This placebo-controlled, double-blind, randomized trial (RAINBOW) was conducted in five European countries to explore the efficacy and safety of CG beta as add-on treatment to follitropin delta in women undergoing ovarian stimulation in a long GnRH agonist protocol. Randomization was stratified by centre and age (30-37 and 38-42 years). The primary endpoint was the number of good-quality blastocysts (Grade 3 BB or higher). Subjects were randomized to receive either placebo or 1, 2, 4, 8 or 12 µg CG beta added to the daily individualized follitropin delta dose during ovarian stimulation. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 620 women (30-42 years) with anti-Müllerian hormone (AMH) levels between 5 and 35 pmol/l were randomized in equal proportions to the six treatment groups and 619 subjects started treatment. All 619 subjects were treated with an individualized dose of follitropin delta determined based on AMH (Elecsys AMH Plus Immunoassay) and body weight. Triggering with rhCG was performed when 3 follicles were ≥17 mm but no more than 25 follicles ≥12 mm were reached. MAIN RESULTS AND THE ROLE OF CHANCE: The demographic characteristics were comparable between the six treatment groups and the overall mean age, body weight and AMH were 35.6 ± 3.3 years, 65.3 ± 10.7 kg and 15.3 ± 7.0 pmol/l, respectively. The incidence of cycle cancellation (range 0-2.9%), total follitropin delta dose (mean 112 µg) and duration of stimulation (mean 10 days) were similar across the groups. At stimulation Day 6, the number and size of follicles was similar between the treatment groups, whereas at the end-of-stimulation dose-related decrease of the intermediate follicles between 12 and 17 mm was observed in comparison to the placebo group. In contrast, the number of follicles ≥17 mm was similar between the CG beta dose groups and the placebo group. A reduced number of intermediate follicles (12 to 17 mm) and fewer oocytes (mean range 9.7 to 11.2) were observed for all doses of CG beta compared to the follitropin delta only group (mean 12.5). The mean number of good-quality blastocysts was 3.3 in the follitropin delta group and ranged between 2.1 and 3.0 across the CG beta groups. The incidence of transfer cancellation was higher in the 4, 8 and 12 µg group, mostly as no blastocyst was available for transfer. In the group receiving only follitropin delta, the ongoing pregnancy rate (10-11 weeks after transfer) was 43% per started cycle versus 28-39% in CG beta groups and 49% per transfer versus 38-50% in the CG beta groups. There was no apparent effect of CG beta on the incidence of adverse events, which was 48.1% in the placebo group and 39.6-52.3% in the CG beta dose groups. In line with the number of collected oocytes, the overall ovarian hyperstimulation syndrome incidence remained lower following follitropin delta with CG beta (2.0-10.3%) compared with follitropin delta only treatment (11.5%). Regardless of the dose, CG beta was safe and well-tolerated with low risk of immunogenicity. LIMITATIONS, REASONS FOR CAUTION: The effect of the unique glycosylation of CG beta and its associated potency implications in women were not known prior to this trial. Further studies will be needed to evaluate optimal doses of CG beta for this and/or different indications. WIDER IMPLICATIONS OF THE FINDINGS: The high ongoing pregnancy rate in the follitropin delta group supports the use of individualized follitropin delta dosing in a long GnRH agonist protocol. The addition of CG beta reduced the presence of intermediate follicles with the investigated doses and negatively affected all down-stream parameters. Further clinical research will be needed to assess the optimal dose of CG beta in the optimal ratio to follitropin delta to develop this novel combination product containing both FSH and LH activity for ovarian stimulation. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by Ferring Pharmaceuticals, Copenhagen, Denmark. B.M. and P.L. are employees of Ferring Pharmaceuticals. M.F.S., H.V., C.Y.A., M.F., C.B., A.P. and Y.K. have received institutional clinical trial fees from Ferring Pharmaceuticals. C.B. has received payments for lectures from Organon, Ferring Pharmaceuticals, Merck A/S and Abbott. M.F.S. has received payment for lectures from Ferring Pharmaceuticals. Y.K. has received payment for lectures from Merck and travel support from Gedeon Richter. H.V. has received consulting fees from Oxo and Obseva and travel support from Gedeon Richter, Ferring Pharmaceuticals and Merck. C.Y.A. has received payment for lectures from IBSA, Switzerland. M.F and C.Y.A. were reimbursed as members of the Data Monitoring Board in this trial. M.F. has an issued patent about unitary combination of FSH and hCG (EP1633389). TRIAL REGISTRATION NUMBER: 2017-003810-13 (EudraCT Number). TRIAL REGISTRATION DATE: 21 May 2018. DATE OF FIRST PATIENT'S ENROLMENT: 13 June 2018.

Cumulative live birth rate after ovarian stimulation with freeze-all in women with polycystic ovaries: does the polycystic ovary syndrome phenotype have an impact?

RESEARCH QUESTION: Do cumulative live birth rates (CLBR) differ between polycystic ovary syndrome (PCOS) phenotypes when a freeze-all strategy is used to prevent OHSS after ovarian stimulation? DESIGN: A single-centre, retrospective cohort study of 422 women with PCOS or polycystic ovarian morphology (PCOM), in whom a freeze-all strategy was applied after GnRH agonist triggering because of hyper-response in their first or second IVF/ICSI. Primary outcome was CLBR; multivariate logistic regression analysis was used. RESULTS: Phenotype A (hyperandrogenism + ovulation disorder + PCOM [HOP]) (n = 91/422 [21.6%]); phenotype C (hyperandrogenism + PCOM [HP]) (33/422 [7.8%]; phenotype D (ovulation disorder + PCOM [OP]) (n = 161/422 [38.2%]); and PCOM (n = 137/422 [32.5%]. Unadjusted CLBR was similar among the groups (69.2%, 69.7%, 79.5% and 67.9%, respectively; P = 0.11). According to multivariate logistic regression analysis, the phenotype did not affect CLBR (OR 0.72, CI 0.24 to 2.14 [phenotype C]; OR 1.55, CI 0.71 to 3.36 [phenotype D]; OR 0.84, CI 0.39 to 1.83 [PCOM]; P = 0.2, with phenotype A as reference). CONCLUSIONS: In women with PCOS, hyper-response after ovarian stimulation confers CLBR of around 70%, irrespective of phenotype, when a freeze-all strategy is used. This contrasts with unfavourable clinical outcomes in women with hyperandrogenism and women with PCOS who underwent mild ovarian stimulation targeting normal ovarian response and fresh embryo transfer. The results should be interpreted with caution because the study is retrospective and cannot be generalized to all cycles as they pertain to those in which hyper-response is observed.