Correlations between clinical parameters, blastocyst morphological classification and embryo euploidy.

OBJECTIVE: The aim of the present study was to evaluate clinical and embryo parameters to predict embryo ploidy. METHODS: In this retrospective analysis, we studied 838 biopsied day-5 blastocysts from 219 patients in the period from May 2021 to July 2022. All embryos were morphologically classified before biopsy and were divided into two groups according to genetic test results. Euploid embryos (299) were compared with aneuploid embryos (539) based on maternal age, anti-Mullerian hormone, antral follicle count, and embryo morphology. RESULTS: Maternal age (36.2±3.0) of euploid embryos was lower than maternal age (37.1±2.5) of aneuploid embryos (p<0.0001). AMH levels were higher (3.9±1.2) in the group of euploid embryos than in the group of aneuploid embryos (3.6±1.3, p<0.0001). However, the AFC was not different in the group of euploid embryos (15.3±6.0) compared to the group of aneuploid embryos (14.5±5.9, p=0.07). The presence of aneuploidy was negatively correlated with top embryo quality (embryos 4AA and 4AB). All euploid embryos (299) were top quality versus 331 of 539 (61.49%) aneuploid embryos (p<0.0001). CONCLUSIONS: We found that euploid embryos were associated with lower maternal age, higher AMH levels, and higher quality embryos.

Serum progesterone level in luteal phase improves pregnancy rate in fresh cycles with blastocyst embryo transfer.

OBJECTIVE: To assess the association between serum level of progesterone during stimulation and in the luteal phase with pregnancy rate in a cohort of patients undergoing in vitro fertilization and embryo transfer (IVF-ET) on day 5. METHODS: Retrospective Cohort Study. Patients: 62 infertile women, aged 24-42 years, undergoing ART at our center from May 2019 to May 2021. Progesterone was evaluated during ovarian stimulation on Day 2, Day 6, and Day 8 of stimulation, day of trigger (P4dhCG), and on the day of blastocyst transfer with 5 days of progesterone supplementation (P4d5+). We also calculated the difference of P4d5+ with P4dhCG. (∆P4). Then we divided the patients into two groups based on progesterone serum levels at P4d5+; <10ng/ml (Group A), ≥10ng/ml (Group B). The Student's t-test was performed for continuous variables; Mann-Whitney's Test and Spearman's Test were used where appropriate for categorical variables. p<0.05 was considered statistically significant. RESULTS: There were positive correlations between ßhCG positive with P4d5+ (p<0.001; Rho 0.770) and ∆P4 (p<0.001; Rho 0.703). The pregnancy rate doubled when the serum progesterone level was ≥10ng/ml on the fifth day of progesterone supplementation compared with P4<10ng/ml (44% vs. 21%, respectively). CONCLUSIONS: The pregnancy rate was positively correlated with the serum P4 level on the fifth day of progesterone supplementation and with the difference between the serum progesterone level in the Dd5+ / dhCG. A higher pregnancy rate was observed when serum progesterone level on the fifth day of progesterone supplementation was ≥10ng/ml.

Utilization of in vitro maturation in cases with a FSH receptor mutation.

PURPOSE: To identify the FSH receptor (FSHR) variant and efficacy of in vitro maturation (IVM) in a 28-year-old woman with secondary amenorrhea, primary infertility, and ovarian resistance to FSH, and to analyze the genotype-to-phenotype relationship in cases of FSHR mutation for the development of an IVM algorithm for use in patients with gonadotropin resistance syndrome (GRS). METHODS: Oocytes retrieved after menstruation induction with norethisterone, followed by daily estrogen and an ovulatory trigger, underwent IVM, ICSI, and culture in a time-lapse (TL) incubator. Embryo transfers were performed on day 2, and after thawing on day 5. Genes associated with disorders of sex development were sequenced for both the patient and her parents. All reported cases of FSHR mutation were analyzed to investigate genotype/phenotypic relationships. RESULTS: After ovum pickup, seven of 16 oocytes matured and all fertilized. After unsuccessful day 2 transfer, our patient delivered with a thawed day 5 blastocyst, the sole embryo without abnormal TL phenotypes. Genetic analysis revealed a new composite heterozygous FSHR variant. Analysis of our patient case with published cases of GRS revealed associations among FSHR variant genotype, location on the FSHR, functionality of tested variants, and type of amenorrhea. An algorithm for application of IVM for GRS patients was developed. CONCLUSIONS: We report two novel variants of the FSHR. Although IVM successfully matured some oocytes, only one resulted in an embryo with normal TL phenotypes. We recommend FSHR genetic testing in GRS patients, which will help guide their suitability for IVM.

A comparison of the effects of three different estrogen used for endometrium preparation on the outcome of day 5 frozen embryo transfer cycle.

OBJECTIVE: To evaluate the effects of three different estrogen used for endometrium preparation on pregnancy rate, as well as hormone profile on day 5 frozen embryo transfer (FET) cycles. METHODS: Retrospective, observational study. Setting: A tertiary teaching and research private reproductive medicine center. Patients: Ninety patients who were undergoing endometrium preparation for day five frozen embryo transfer cycle (FET). Intervention(s): The women were divided in three groups according to the administration route of estrogen (E2): oral (Primogyna), transdermal patches (Estradot), or transdermal gel (Oestrogel Pump). These administration routines of estrogen are equivalent to 6mg of estradiol daily. All women received 600mg of vaginal progesterone (P) per day (Utrogestan) for luteal phase support. We drew blood samples on starting P day, as well as on beta hCG day for E2 and P measurements. Main Outcome Measure(s): Clinical pregnancy rates (PR). RESULTS: Patient features in the three groups were comparable. There were no significant differences concerning implantation rate, clinical PR, miscarriage rate, multiple-pregnancy rate, or E2 and P levels on starting P day and on beta hCG day. CONCLUSIONS: In FET cycles with oral (Primogyna) or transdermal patches (Estradot), or transdermal gel (Oestrogel Pump), there was no significant difference on pregnancy rates.

Which ovarian reserve marker relates to embryo quality on day 3 and blastocyst; age, AFC, AMH?

OBJECTIVE: The aim of the present prospective study was to evaluate which ovarian reserve marker would be more reliable as the quality of the A + B embryos (day 3 and blastocyst). METHODS: We ran a prospective study with 124 infertile women, aged 24-48 years, from 2017 to 2018. The patients were divided into 3 groups according to age and the subgroups were compared for AMH, AFC, number of A+B embryos. New division of the 3 groups was performed based on the AMH, and the subgroups were compared for age, AFC and number of A+B embryos. Finally, we divided the patients into 3 groups, based on the AFC, and we compared the subgroups for age, AMH and number of A+B embryos. P<0.05 was considered statistically significant. RESULTS: When the 124 patients were divided according to age, we found a significant fall in an A+B embryo quality (day3; blastocyst) after 35 years (p<0.038; p<0.035), and more severely after 37 years (p<0.032; p<0.027). When the 124 patients were divided according to AMH, there was a significant fall in A+B embryo quality (day 3; blastocyst), with AMH<1ng/ml (p<0.023; p<0.021). When the 124 patients were divided according to AFC, there was a significant fall in A+B embryo quality (day 3; blastocyst) with AFC<7 (p<0.025; p<0.023). These markers had significant associations with embryo quality (p<0.005). CONCLUSION: Age, AFC and AMH have significant associations with A +B embryo quality on day 3 and blastocyst.

How time to healthy singleton delivery could affect decision-making during infertility treatment: a Delphi consensus.

RESEARCH QUESTION: How might time to healthy singleton delivery affect decision-making during infertility treatment? DESIGN: This was a Delphi consensus investigating expert opinion that comprised three steps. In Step 1, 12 experts developed statements. In Step 2, 27 experts (including 12 from Step 1) voted (online survey) on their agreement/disagreement with each statement (providing reasons). Consensus was reached if ≥66% of participants agreed/disagreed. Statements not reaching consensus were revised and the process repeated until consensus was achieved. In Step 3 details of the final agreed statements were communicated. RESULTS: Twelve statements were developed, and consensus (agreement) was reached on all after one round of voting. CONCLUSIONS: Time to healthy singleton delivery should be taken into consideration when making decisions related to infertility treatment, and it is important that fertility treatment is provided in a timely manner, avoiding over- or under-treatment. In all subfertile women <40 years old, IVF outcomes could be optimized by performing up to six single-embryo transfers and certain procedures might reduce time to healthy singleton delivery. These procedures include preimplantation genetic testing for aneuploidies, frozen replacement cycles immediately after failed fresh cycles and use of gonadotrophin-releasing hormone antagonists. Finally, the number of oocytes retrieved should be maximized to increase cumulative live birth rate.

Introduction: Continuing story of premature progesterone elevation.

Since the late 1980s premature progesterone elevation has repeatedly caught the attention of investigators, not only for its importance, but also because studies have shown differing results and conclusions, constituting an unexplainable sequence of doubts and uncertainties. This issue's Views and Reviews section seeks to present a sequence of short and complementary papers summarizing the whole story and updating the reader on the current information and perspectives on premature progesterone elevation.

Premature progesterone elevation in controlled ovarian stimulation: to make a long story short.

Over the past decades many of us have contributed to the controversy surrounding the origins and consequences of premature progesterone elevation during controlled ovarian stimulation. In this article, we attempt to retrace the progression of information on this complex subject which required reviewing a number of publications that often contradicted one another. The definition of premature progesterone elevation, the pathophysiological mechanisms underlying the high peripheral progesterone levels, and the debated consequences of this event on in vitro fertilixation-embryo transfer outcome will be addressed from a historical perspective.

Serum antimüllerian hormone levels are independently related to miscarriage rates after in vitro fertilization-embryo transfer.

OBJECTIVE: To investigate whether serum antimüllerian hormone (AMH) levels are independently related to miscarriage rates after in vitro fertilization-embryo transfer (IVF-ET). DESIGN: Cohort study. SETTING: University-affiliated IVF-ET center. PATIENT(S): A total of 1,060 patients who attained a clinical pregnancy after IVF-ET. INTERVENTIONS(S): Centralized serum AMH measurements were performed within the 12 months before IVF-ET. Binary logistic regression was used to verify whether serum AMH levels were associated with the occurrence of a miscarriage independently from confounding factors, such as age and intensity of ovarian response to controlled ovarian stimulation assessed by the number of oocytes retrieved. MAIN OUTCOME MEASURE(S): Miscarriage rates. RESULT(S): In patients displaying reduced serum AMH levels, miscarriage rates were significantly increased independently from age and the number of oocytes retrieved. CONCLUSION(S): The present data indicate that serum AMH levels are independently associated with the occurrence of a miscarriage after IVF-ET.

Non-equivalence of anti-Müllerian hormone automated assays-clinical implications for use as a companion diagnostic for individualised gonadotrophin dosing.

STUDY QUESTION: Can anti-Müllerian hormone (AMH) automated immunoassays (Elecsys® and Access) be used interchangeably as a companion diagnostic for individualisation of follitropin delta dosing? SUMMARY ANSWER: The Access assay gives systematically higher AMH values than the Elecsys® assay which results in over 29% of women being misclassified to a different follitropin delta dose. WHAT IS KNOWN ALREADY: Follitropin delta is the first gonadotrophin to be licenced with a companion diagnostic, the Roche Elecsys® AMH Plus assay. Alternative automated AMH assays including the Beckman Coulter Access immunoassay are considered to provide similar results, but clarification of their suitability as an off-licence companion diagnostic for follitropin delta is required. STUDY DESIGN, SIZE, DURATION: We systematically searched the existing literature for studies that had measured AMH using both automated assays in the same cohort of women. Individual paired patient data were acquired from each author and combined with unpublished data. PARTICIPANTS/MATERIALS, SETTING, METHODS: We identified five eligible prospective published studies and one additional unpublished study. A 100% response from the authors was achieved. We collected paired AMH data on samples from 848 women. Passing-Bablok regression and Bland-Altman plots were used to compare the analytical performance of the two assays. The degree of misclassification to different treatment categories was estimated should the Access AMH be used as a companion diagnostic instead of the Elecsys AMH in determining the dosing of follitropin delta. MAIN RESULTS AND THE ROLE OF CHANCE: The Passing-Bablok regression shows a linear relationship (Access = -0.05 + 1.10 × Elecsys). The Access assay systematically gave higher values by an average of 10% compared with the Elecsys assay (slope = 1.10, 95% CI: 1.09 to 1.12). The average of the difference between the two assays was 2.7 pmol/l. The 95% limits of agreement were -11.7 to 6.3. Overall 253 (29.3%) women would have received an inappropriate follitropin delta dose if the Beckman Coulter Access assay was used. Specifically, a substantial proportion of women (ranging from 49% to 90% depending on the AMH category) would receive a lower dose of follitropin delta based on the Access AMH assay. Up to 10% (ranging from 2.5% to 10%) of women with high ovarian reserve would have been misclassified to a greater dose of follitropin delta based on the Access AMH assay. LIMITATIONS REASONS FOR CAUTION: We compared the values of the two principal automated assays, extrapolation of our findings to other automated AMH assays would require similar comprehensive examination. WIDER IMPLICATIONS OF THE FINDINGS: An international standard for the calibration of the automated AMH assays is warranted to facilitate efficient use of AMH as a companion diagnostic. The variable calibration of alternative automated AMH assays may adversely impact on the performance of the follitropin delta dosing algorithm. STUDY FUNDING/COMPETING INTEREST(S): No formal funding has been received for this study. SI is funded by a UK Medical Research Council skills development fellowship (MR/N015177/1). SMN has received speakers fees, travel to meetings and participated in advisory Boards for Beckman Coulter, IBSA, Ferring Pharmaecuticals, Finox, Merck Serono, Merck and Roche Diagnostics. SMN has received research support from Ansh laboratories, Beckman Coulter, Ferring Pharmaceuticals and Roche Diagnostics. TRIAL REGISTRATION NUMBER: N/A.

Dysregulation of the Anti-Müllerian Hormone System by Steroids in Women With Polycystic Ovary Syndrome.

Context: Anti-Müllerian hormone (AMH) and AMH type II receptor (AMHR2) are overexpressed in granulosa cells (GCs) from women with polycystic ovary syndrome (PCOS), the most common cause of female infertility. Objective: The aim of the study was to compare the regulation of the AMH/AMHR2 system by 5α-dihydrotestosterone (5α-DHT) and estradiol (E2) in GCs from control subjects and women with PCOS. Design, Setting, Patients: Experiments were performed on follicular fluids (FF) and GCs from women undergoing in vitro fertilization. Main Outcome Measures: FF steroid levels were measured by mass spectrometry, and messenger RNA (mRNA) accumulation was quantified by reverse transcription real-time polymerase chain reaction. Results: Total testosterone (T), free T, and 5α-DHT FF levels were significantly higher (P < 0.001) in women with PCOS than in controls. However, E2 and sex hormone-binding globulin concentrations were comparable between the two groups. In GCs from control women, the AMH and AMHR2 expression were not affected by 5α-DHT treatment, whereas AMH mRNA levels were upregulated by 5α-DHT in GCs from patients with PCOS (2.3-fold, P < 0.01) overexpressing the androgen receptor (1.4-fold, P < 0.05). E2 downregulated the AMH and AMHR2 expression in GCs from control women (1.4-fold, P < 0.001 and 1.8-fold, P < 0.01, respectively) but had no effect on these genes in GCs from women with PCOS. This differential effect of E2 was associated with a higher estrogen receptor 1 expression in GCs from women with PCOS (1.9-fold, P < 0.05). Conclusions: In GCs from women with PCOS, the regulation of AMH and AMHR2 expression is altered in a way that promotes the overexpression of the AMH/AMHR2 system, and could contribute to the follicular arrest observed in these patients.

Steroid hormone pretreatments in assisted reproductive technology.

Adequate availability and FSH sensitivity of ovarian antral follicles and coordination of their growth during controlled ovarian hyperstimulation (COH) rank among factors that may determine outcome, particularly in patients presenting ovarian function defects and so-called "poor responders." Growing evidence indicates that both factors are positively influenced by steroid hormone pretreatments. First, data from studies conducted in both animals and in women exposed to virilizing androgen doses indicate that androgen pretreatments may increase follicle responsiveness to FSH and/or the number of growing follicles in the ovary, thereby constituting an interesting perspective in the management of "poor responders." Second, overcoming pre-COH heterogeneities in antral follicle sizes, which are more pronounced in "poor responders," to achieve adequate coordination of multiple follicular growth during COH also is contributive. For this, suppression or attenuation of the premature FSH increase during the preceding late luteal phase using sex steroid pretreatments (oral contraceptives, synthetic progestogens, or estradiol), or additional strategies such as premenstrual GnRH antagonist administration has been shown to be effective. The present paper will critically review proposed mechanisms and clinical results of sex steroid hormone pretreatments in these two different indications as an effort to optimizing COH outcome.

New automated antimüllerian hormone assays are more reliable than the manual assay in patients with reduced antral follicle count.

OBJECTIVE: To compare the strength of the relationship between antral follicle count (AFC) and serum antimüllerian hormone (AMH) concentrations obtained with two automated and one manual AMH assays in three different AFC populations. DESIGN: Prospective cohort study. SETTING: University-affiliated IVF-ET center. PATIENT(S): Frozen-thawed serum samples of 211 assisted conception candidates, aged 24-43 years. INTERVENTION(S): Serum AMH was measured using one manual (AMH Gen II) and two fully automated (Access AMH and Elecsys AMH) assays. Antral follicle count was performed under strictly standardized conditions and sorted into three groups according to tercile values: low AFC (3-12 follicles; n = 73), intermediate AFC (13-20 follicles; n = 65), and high AFC (21-84 follicles; n = 73). MAIN OUTCOME MEASURE(S): Strength of correlation between AMH levels and AFC. RESULT(S): Overall, AMH levels were lower with Access AMH (-16%) and Elecsys AMH (-20%) than with AMH Gen II. Remarkably, the strength of correlations between AFC and circulating AMH levels was the same with the three assays (r = 0.83). Yet in the low AFC group, serum AMH levels obtained by Access AMH and Elecsys AMH showed a stronger correlation with AFC (r = 0.63 and r = 0.65, respectively) than the AMH Gen II (r = 0.52), a phenomenon that was not observed in the remaining AFC groups. CONCLUSION(S): As compared with conventional AMH Gen II assay results, [1] serum AMH concentrations were -16% and -20% lower with Access AMH and Elecsys AMH, respectively; and [2] automated assays were more strongly correlated to AFC in the subset of patients with reduced follicle count.

Most Cleaved Anti-Müllerian Hormone Binds Its Receptor in Human Follicular Fluid but Little Is Competent in Serum.

CONTEXT: Anti-Müllerian hormone (AMH) is an important clinical marker for diagnosing and assessing the reproductive status and/or disorders in men and women. Most studies have not distinguished between levels of inactive AMH precursor and the cleaved noncovalent complex that binds the AMH type II receptor (AMHRII) and initiates signaling. OBJECTIVE: The objective of the study was to measure the levels of AMH cleavage and bioactivity in human body fluids. DESIGN, SETTING, AND PATIENTS: AMH cleavage levels and bioactivity were measured in the serum of six boys and in the follicular fluid and serum of nine control women and 13 women with the polycystic ovary syndrome (PCOS). MAIN OUTCOME MEASURES: AMH cleavage levels were measured by capturing AMH with an anti-AMH antibody, followed by Western blotting. The bioactivity of cleaved AMH was assessed with an ELISA that measures the levels of AMH capable of binding AMHRII. RESULTS: PCOS women have an elevated level of AMH cleavage in their follicular fluid (24% vs 8% in control women), and most of the cleaved AMH can bind AMHRII. Higher levels of cleavage are observed in female (60%) and male (79%) serum, but very little of the cleaved AMH can bind AMHRII. CONCLUSIONS: These results support an autocrine role for AMH in the pathophysiology of PCOS in the follicle. In addition, they indicate that AMH undergoes interactions or structural changes after cleavage that prevent receptor binding, meaning, unexpectedly, that the level of cleaved AMH in biological fluids does not always reflect the level of bioactive AMH.