STUDY QUESTION Does letrozole co-treatment during ovarian stimulation with gonadotrophins for IVF reduce the proportion of women with premature progesterone levels above 1.5 ng/ml at the time of triggering final oocyte maturation? SUMMARY ANSWER The proportion of women with premature progesterone above 1.5 ng/ml was not significantly affected by letrozole co-treatment. WHAT IS KNOWN ALREADY IVF creates multiple follicles with supraphysiological levels of sex steroids interrupting the endocrine milieu and affects the window of implantation. Letrozole is an effective aromatase inhibitor, normalizing serum oestradiol, thereby ameliorating some of the detrimental effects of IVF treatment. STUDY DESIGN, SIZE, DURATION A randomized, double-blinded placebo-controlled trial investigated letrozole intervention during stimulation for IVF with FSH. The trial was conducted at four fertility clinics at University Hospitals in Denmark from August 2016 to November 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS A cohort of 129 women with expected normal ovarian reserve (anti-Müllerian hormone 8–32 nmol/l) completed an IVF cycle with fresh embryo transfer and received co-treatment with either 5 mg/day letrozole (n = 67) or placebo (n = 62), along with the FSH. Progesterone, oestradiol, FSH, LH and androgens were analysed in repeated serum samples collected from the start of the stimulation to the mid-luteal phase. In addition, the effect of letrozole on reproductive outcomes, total FSH consumption and adverse events were assessed. MAIN RESULTS AND THE ROLE OF CHANCE The proportion of women with premature progesterone >1.5 ng/ml was similar (6% vs 0% (OR 0.0, 95% CI [0.0; 1.6], P = 0.12) in the letrozole versus placebo groups, respectively), whereas the proportion of women with mid-luteal progesterone >30 ng/ml was significantly increased in the letrozole group: (59% vs 31% (OR 3.3, 95% CI [1.4; 7.1], P = 0.005)). Letrozole versus placebo decreased oestradiol levels on the ovulation trigger day by 68% (95% CI [60%; 75%], P < 0.0001). Other hormonal profiles, measured as AUC, showed the following results. The increase in LH in the letrozole group versus placebo group was 38% (95% CI [21%; 58%], P < 0.0001) and 34% (95% CI [11%; 61%], P = 0.006) in the follicular and luteal phases, respectively. In the letrozole group versus placebo group, testosterone increased by 79% (95% CI [55%; 105%], P < 0.0001) and 49% (95% CI [30%; 72%], P < 0.0001) in the follicular and luteal phases, respectively. In the letrozole group versus placebo group, the increase in androstenedione was by 85% (95% CI [59%; 114%], P < 0.0001) and 69% (95% CI [48%; 94%], P < 0.0001) in the follicular and luteal phases, respectively. The ongoing pregnancy rate was similar between the letrozole and placebo groups (31% vs 39% (risk-difference of 8%, 95% CI [−25%; 11%], P = 0.55)). No serious adverse reactions were recorded in either group. The total duration of exogenous FSH stimulation was 1 day shorter in the intervention group, significantly reducing total FSH consumption (mean difference −100 IU, 95% CI [−192; −21], P = 0.03). LIMITATIONS, REASONS FOR CAUTION Late follicular progesterone samples were collected on the day before and day of ovulation triggering for patient logistic considerations, and the recently emerged knowledge about diurnal variation of progesterone was not taken into account. The study was powered to detect hormonal variations but not differences in pregnancy outcomes. WIDER IMPLICATIONS OF THE FINDINGS Although the use of letrozole has no effect on the primary outcome, the number of women with a premature increase in progesterone on the day of ovulation triggering, the increased progesterone in the mid-luteal phase due to letrozole may contribute to optimizing the luteal phase endocrinology. The effect of letrozole on increasing androgens and reducing FSH consumption may be used in poor responders. However, the effect of letrozole on implantation and ongoing pregnancy rates should be evaluated in a meta-analysis or larger randomized controlled trial (RCT). STUDY FUNDING/COMPETING INTEREST(S) Funding was received from EU Interreg for ReproUnion and Ferring Pharmaceuticals, and Roche Diagnostics contributed with assays. N.S.M. and A.P. have received grants from Ferring, Merck Serono, Anecova and Gedeon Richter, and/or personal fees from IBSA, Vivoplex, ArtPred and SPD, outside the submitted work. The remaining authors have no competing interests. TRIAL REGISTRATION NUMBERS NCT02939898 and NCT02946684 TRIAL REGISTRATION DATE 15 August 2016. DATE OF FIRST PATIENT’S ENROLMENT 22 August 2016.

STUDY QUESTION What are the downstream endocrine and paracrine consequences of letrozole (LZ) cotreatment during ovarian stimulation and is follicle growth and recruitment affected? SUMMARY ANSWER Letrozole cotreatment induces marked changes in both the follicular and luteal phase endocrinology causing potentiation of follicle diameter and an improved corpus luteum function without affecting the secondarily recruited follicle cohort. WHAT IS KNOWN ALREADY Letrozole is a third-generation aromatase inhibitor that is well-established as an effective ovulatory agent, while its possible benefits in standard in vitro fertilization protocols are less thoroughly investigated. STUDY DESIGN, SIZE, DURATION This study included a double-blinded, placebo-controlled, randomized study with LZ or placebo intervention during ovarian stimulation for IVF treatment, an observational preceding baseline natural cycle and a succeeding follow-up visit. Participants were enrolled between August 2016 and November 2018. Data from the randomized, stimulated cycle were part of a larger RCT, which was previously published. PARTICIPANTS/MATERIALS, SETTING, METHODS The study was conducted at a public fertility clinic at Herlev Hospital, Denmark, including 31 healthy, normo-responding women eligible for IVF treatment. They underwent a natural baseline cycle and were subsequently randomized to receive either LZ 5 mg (n = 16) or placebo (n = 15) daily during ovarian stimulation from cycle day (CD) 2–3 until induction of ovulation. Throughout both cycles, monitoring was performed every third day with transvaginal ultrasound for assessment of follicle count and diameter, and blood analyses for the determination of twelve endocrine and paracrine parameters. A follow-up assessment was performed at CD2–3 in the succeeding cycle. In the randomized part of the study, we determined differences in blood parameters, follicle recruitment, and follicle diameter. In the observational part of the study, we assessed follicle recruitment in between cycles and its correlation to endocrine parameters. MAIN RESULTS AND THE ROLE OF CHANCE Letrozole cotreatment significantly suppressed oestradiol (E2) concentrations in the follicular phase (area under the curve (AUC) −58% (95% CI [−70%; −43%], P < 0.001)) and luteal phase (AUC −39% [−63%; −1%], P = 0.046). This had a marked effect on the endocrine and paracrine output with increased follicular phase luteinizing hormone (AUC +37% [3%; 82%], P = 0.033), androstenedione (AUC +36% [6%; 74%], P = 0.016), testosterone (AUC +37% [7%; 73%], P = 0.013) and 17-OH-progesterone (AUC +114% [10%; 318%], P = 0.027). Furthermore, follicle-stimulating hormone (FSH) was increased at stimulation day 5 in the LZ group (P < 0.05). In the luteal phase, increased corpus luteum output was reflected by elevated progesterone (AUC +44% [1%; 104%], P = 0.043), inhibin A (AUC +52% [11%; 108%], P = 0.011), androstenedione (AUC +31% [9%; 58%], P = 0.006) and testosterone (AUC +29% [6%; 57%], P = 0.012) in the LZ group. The altered balance between oestrogens and androgens was reflected in a markedly reduced SHBG concentration in the LZ group throughout the luteal phase (AUC −35% [−52%; −11%], P = 0.009). Endocrine and paracrine parameters were similar between groups at the follow-up visit. Letrozole cotreatment significantly increased the mean number of follicles >16 mm at oocyte retrieval (7.2 vs 5.2, difference: 2.0, 95% CI [0.1; 3.8], P = 0.036), while the mean total number of follicles at oocyte retrieval was the same (23.7 vs 23.5, difference: 0.2 [−5.8; 6.1], P = 0.958), and the mean FSH consumption during the stimulated cycle was similar (1500 vs 1520 IU, difference −20 IU [−175; 136], P = 0.794). Between cycles, the mean antral follicle count at CD2–3 was unchanged (natural cycle 19.0, stimulated cycle 20.9, follow-up cycle 19.7, P = 0.692) and there was no effect of LZ cotreatment on the recruitment of the next follicle cohort (test for interaction, P = 0.821). LIMITATIONS, REASONS FOR CAUTION This study included a relatively small, selected group of healthy women with an expected normal ovarian function and reserve, and the effects of LZ may therefore be different in other patient groups. WIDER IMPLICATIONS OF THE FINDINGS We confirm some previous findings concerning increased follicle growth and increased endogenous FSH and androgen production, which support the rationale for further studies on the use of LZ cotreatment, for example, as a form of endogenous androgen priming sensitizing the follicle to FSH. Letrozole appears to improve the luteal phase with better stimulation of corpus luteum and progesterone secretion. STUDY FUNDING/COMPETING INTEREST(s) The authors declare no conflicts of interest relating to the present work. TRIAL REGISTRATION NUMBER NCT02939898.

STUDY QUESTION Does adjuvant letrozole in ovarian stimulation for in vitro fertilization (IVF) decrease the uterine peristalsis frequency (UPF) prior to fresh embryo transfer (ET)? SUMMARY ANSWER Adjuvant letrozole in ovarian stimulation for IVF does not reduce the UPF significantly prior to fresh ET. WHAT IS KNOWN ALREADY Throughout the cycle uterine peristalsis aids spermatozoa transport to the fallopian tube and may affect implantation. At fresh ET, UPF is negatively correlated with implantation and clinical pregnancy rates and is believed to be modulated by estradiol and progesterone. High levels of estradiol, from multiple follicular development, in ovarian stimulation have been reported to increase UPF, whereas progesterone is considered to be an utero-relaxant. The influence of androgens is unclear. Co-treatment with letrozole during gonadotropin ovarian stimulation limits the supra-physiological estradiol rise and may therefore reduce UPF prior to fresh ET. STUDY DESIGN, SIZE, DURATION This study was carried out on subjects participating in a single centre double blinded randomised controlled trial (RCT) of the impact of letrozole on follicle development and endocrine profiles, and investigated the impact of adjuvant letrozole in ovarian stimulation for IVF on UPF prior to fresh ET and the correlations of UPF with endocrine markers. Between 2016 and 2017, 39 women expected to be normal responders were randomised to co-treatment with letrozole or placebo. Of these, 33 women completed this element of the study. The study was carried out according to the Helsinki Declaration and the ICH-Good-Clinical-Practice. PARTICIPANTS/MATERIALS, SETTING, METHODS Eligible women were randomised 1:1 to adjuvant treatment with letrozole 5 mg/day or placebo in an antagonist protocol using a fixed dose of recombinant (r) FSH 150 IU/day. Final maturation was triggered with hCG 6,500 IU and luteal support with vaginal progesterone was administered from the day following oocyte aspiration. Less than one hour prior to fresh ET, six-minute duration transvaginal ultrasound recordings of the uterus in sagittal section were performed and blood samples were drawn. MAIN RESULTS AND THE ROLE OF CHANGE A total of 33 women completed the study (letrozole n = 17; placebo n = 16). Age, BMI and ovarian reserve markers were similar between the groups. On day of ET, serum estradiol levels were significantly suppressed in the letrozole group to a mean of 867 ± 827 pmol/L compared to 3,110 ± 1,528 pmol/L in the placebo group (P < 0.001). Mean UPF prior to fresh ET did not differ between the intervention and placebo group (3.3 ± 0.36 versus 3.5 ± 0.51 per minute respectively, P = 0.108). UPF was assessed and agreed by two observers who were blinded to adjuvant treatment. Two patients were excluded due to poor quality of the ultrasound recordings. Supra-physiological serum estradiol in the placebo group were negatively correlated with UPF (P = 0.014; R = -0.62), but the more physiological serum estradiol levels in the letrozole group showed no correlation with UPF (P = 0.567; R = 0.15). Serum progesterone levels were similar in both groups and did not show any significant correlation with UPF. Testosterone levels were significantly higher in the letrozole group (P = 0.005) and showed a non-significant trend that negatively correlated with UPF in the placebo group (P-value=0.071, R= -0.48). LIMITATIONS, REASONS FOR CAUTION Limitations of the study included the limited sample size and the lack of a power calculation specifically determined for this endpoint. WIDER IMPLICATIONS OF THE FINDINGS The supra-physiological levels of estradiol generated during ovarian stimulation were significantly suppressed in the intervention group. However, UPF prior to fresh ET was similar in both groups. Modulating the luteal phase sex steroids with adjuvant letrozole had little measured impact on UPF. Any beneficial effect of adjuvant letrozole during ovarian stimulation is unlikely to be due to significant modulation of UPF. STUDY FUNDING/COMPETING INTEREST(S) MDH's salary was funded by an unrestricted research grant from Gedeon Richter. The expenses of the study was funded by a scientific collaboration: ReproUnion, co-financed by the European Union, Interreg Öresund-Kattegat-Skagerrak and Ferring Pharmaceuticals. The assays for the analyses were funded by Roche Diagnostics and an unrestricted research grant from Merck Life Science AS, Denmark. The authors have no competing interests to declare regarding this study. TRIAL REGISTRATION NUMBER Clinicatrials.gov: NCT02939898, EudraCT no.: 2015-005683-41.

Study question Does letrozole co-treatment during ovarian stimulation for in vitro fertilization (IVF) impact oocyte yield, embryo development, and live birth rate (LBR) in expected normal responders? Summary answer Letrozole co-treatment leads to similar oocyte yield, embryo- development and utilisation rate, and live birth rate after transfer in the fresh cycle. What is known already Letrozole reduces oestradiol levels by inhibiting the aromatization of androgens, this diminishes pituitary suppression and increases endogenous gonadotrophin release. These effects are utilized in ovulation induction in PCOS patients and may benefit certain patients during IVF treatment. In addition, we have recently reported in an RCT that co-treatment with letrozole increases luteal phase progesterone levels and, consistent with previous studies, no significant effect on pregnancy rates was observed. However, the impact of the changes in the endocrine milieu caused by letrozole co-treatment on the developing oocytes and embryos remains unclear as most published studies are retrospective and lack appropriate control-groups. Study design, size, duration A multicentre double-blinded randomised placebo-controlled trial was conducted in four fertility clinics at University Hospitals in Denmark from August 2016 to November 2018. 159 women were randomised, 129 women received single embryo (cleavage stage or blastocyst) transfer in the fresh cycle, and the surplus 257 embryos were vitrified. The study was conducted following the Helsinki Declaration and the ICH-Good-Clinical-Practice. Data collection and reporting followed the guidelines of CONSORT to achieve transparent reporting of trials. Participants/materials, setting, methods Women with expected normal ovarian response received an antagonist protocol with fixed-dose FSH. Co-treatment consisted of 5mg letrozole or placebo from the start of stimulation until the day of triggering final oocyte maturation with hCG. Standardized morphological evaluation was performed at all centres (applying the Istanbul consensus and Gardner-score). A sub-cohort of the embryos were cultured in an EmbryoScope incubator and further assessed by morphokinetic annotation and reported with.KIDScoreD3. Ongoing pregnancies were followed until birth. Main results and the role of chance A total of 1268 oocytes were retrieved from 154 women, leading to development of 386 usable embryos that were either transferred or vitrified. Utilisation rate (the proportion of usable embryos to oocytes) was similar in the letrozole group and placebo group with 0.31 vs. 0.36 (mean difference (MD): -0.05, 95% CI[-0.03;0.12], P = 0.25). Morphologically grading the embryos as “good”, “fair”, and “poor” quality showed similar results, the odds of having a higher quality were 0.78 times higher in the letrozole- vs. placebo group (CI[0.5;1.1], P = 0.2). Morphokinetic annotations (n = 302 embryos) showed similar results with the odds of having a high KIDScore3 1.3 times higher in the letrozole – vs. placebo group (CI[0.9;2.1], P = 0.2). The endometrial thickness at transfer was thinner in the letrozole compared to the placebo group with 8.5 vs. 9.5 mm (MD 1.0, 95% CI[0.4;1.6], P = 0.001) but with the same ultrasonic echogenicity and a preovulatory three-layer appearance. After embryo transfer in the fresh cycle (n = 129), no differences were seen in the pregnancy outcomes: positive serum hCG, ongoing pregnancy rate, early miscarriage rate, foetal miscarriage rate, or LBR. LBR per initiated cycle were similar as well (n = 159): 23.8% vs. 29.1% (MD of -5,3%, CI[-20.3%;9.6%], P = 0.48) in letrozole vs. placebo group, respectively. Limitations, reasons for caution Four clinics participating could lead to interobserver variability. However, bias was reduced by block-randomised design. All data are derived from an RCT, the primary endpoint of which, preovulatory progesterone, was the basis of the sample size calculation. The study may have been underpowered to show a significant impact on LBR. Wider implications of the findings Letrozole co-treatment during ovarian stimulation with gonadotrophins did not have an impact on the oocyte yield and embryo development. These data suggest that letrozole co-treatment is unlikely to compromise outcomes in its growing indications: ovulation induction, IVF in poor responders, and fertility preservation in breast cancer patients. Trial registration number NCT02946684

Study question Does adjuvant letrozole in ovarian stimulation (OS) for in vitro fertilization (IVF) decrease the uterine peristalsis frequency (UPF) prior to fresh embryo transfer (ET)? Summary answer Adjuvant letrozole in (OS) for IVF does not reduce the UPF significantly prior to fresh ET. What is known already Throughout the cycle UPF aids spermatozoa transport to the fallopian tube and may affect implantation. At fresh, ET UPF is negatively correlated with implantation- and clinical pregnancy rates and is believed to be modulated by estradiol and progesterone. High levels of estradiol, from multiple follicular development, in OS have been reported to increase UPF, whereas progesterone is considered to be utero-relaxant. The influence of androgens is unclear. Co-treatment with letrozole during gonadotropin OS limits the estradiol rise the supra-physiological estradiol and may therefore reduce UPF prior to fresh ET. Study design, size, duration: This single centre study was nested within a multicentre double blinded RCT investigating the impact of letrozole co-treatment during gonadotropin OS for IVF on late follicular and luteal estradiol, progesterone and testosterone levels. Between 2016 and 2017, 39 women expected normal responders were randomised to co-treatment with letrozole or placebo. Of these, 33 women completed this element of the study. The study was carried out according to the Helsinki Declaration and the ICH-Good-Clinical-Practice. Participants/materials, setting, methods Eligible women were randomised 1:1 to adjuvant treatment with letrozole 5 mg/day or placebo in an antagonist protocol using a fixed dose of recFSH 150 IU/day. Final maturation was triggered with rhCG 6,500 IU and luteal support with vaginal progesterone was administered from the day following oocyte aspiration. Less than one hour prior to fresh ET, six minute duration transvaginal ultrasound recordings of the uterus in sagittal section were performed and blood samples were drawn. Main results and the role of chance A total of 33 women completed the study (letrozole n = 17; placebo n = 16). Age, BMI, and ovarian reserve markers were similar between the groups. On day of ET, serum estradiol levels were significantly suppressed in the letrozole group to mean 867 ± 827 pmol/L compared to 3,110 ± 1,528 pmol/L in the placebo group (P < 0.0001). Mean UPF prior to fresh ET did not differ between the intervention and control group (3.3 ± 0.36 versus 3.5 ± 0.51 per minute respectively, P = 0.108). UPF was assessed and agreed by two observers who were blind to adjuvant treatment. Two patients were excluded due to poor quality of the ultra sound recording. Supra-physiological serum estradiol in the placebo group was negatively correlated with UPF (P = 0.014; R = –0.62), but the more physiological serum estradiol levels in the letrozole group showed no correlation with UPF (P = 0.567; R = 0.15). Serum progesterone levels were similar in both groups and did not show any significant correlation with UPF. Testosterone levels were significantly higher in the letrozole group (P = 0.005) and showed a non-significant trend negatively correlated with UPF in the placebo group (P-value=0.07, R= –0.48). Limitations, reasons for caution The limited sample size risks masking minor effects. Wider implications of the findings: The supra-physiological levels of estradiol were significantly supressed in the intervention group, but UPF prior to fresh ET was similar in both groups. UPF is not strongly correlated to luteal phase sex steroid levels. Any beneficial effect of adjuvant letrozole during OS is not through an impact of UPF. Trial registration number NCT02939898

Study question What are the endocrine and paracrine effects of letrozole (LZ) co-treatment during ovarian stimulation (OS) and is follicle growth and recruitment affected? Summary answer Letrozole induces marked changes in the follicular and luteal phase endocrinology causing potentiation of follicle growth and corpus luteum function without affecting follicle recruitment. What is known already Letrozole is a 3rd generation aromatase inhibitor that is well-established as an effective ovulatory agent, while its possible benefits in standard in vitro fertilization (IVF) protocols are less thoroughly investigated. Study design, size, duration Double-blinded, placebo-controlled, randomized study with LZ or placebo intervention during OS for IVF treatment, a preceding baseline natural cycle, and a succeeding follow-up visit, in total three consecutive cycles. Participants were enrolled between August 2016 and November 2018. Participants/materials, setting, methods Thirty-one healthy, normo-responding women underwent a natural baseline cycle and an antagonist stimulation cycle with either LZ 5 mg/day (n = 16) or placebo (n = 15) during the follicular phase. Every third day, monitoring of follicle count and -growth and blood analyses of 12 endocrine and paracrine parameters were performed. Follicle recruitment was correlated to endocrine parameters, and differences in follicle recruitment, follicle growth and hormone parameters between groups were assessed. Main results and the role of chance In the LZ group, the number of follicles > 16 mm at oocyte pick up was increased (+2.0, 95% CI [0.1;3.8], p = 0.036), while the total number of follicles were the same (+0.2, 95% CI [-5.8;6.1], p = 0.958). The FSH concentration was increased in the LZ group at stimulation day 5 (p < 0.05), though the total FSH consumption during OS was similar (difference: -20 IU, 95% CI [-175;136], p= 0.794). Letrozole co-treatment suppressed oestradiol (E2) concentrations in the follicular phase (area under the curve (AUC) -62%, p < 0.001) and luteal phase (AUC -31%, p = 0.046). We found increased follicular phase LH (AUC +20.5%, p = 0.033), androstenedione (AUC +22%, p = 0.016), testosterone (AUC +33%, p = 0.013), and 17-OH-progesterone (+132%, p = 0.027). Interestingly, the corpus luteum (CL) output in the LZ group was increased with elevated luteal phase progesterone (AUC +35%, p = 0.043), Inhibin A (AUC +67%, p = 0.011), androstenedione (AUC +22%, p = 0.006), and testosterone (AUC +22%, p = 0.012). Hormone parameters were similar between groups at the follow-up visit. There was no effect of LZ co-treatment on recruitment of the next follicle cohort (p = 0.821).  Limitations, reasons for caution This study included a relatively small, selected group of healthy women with an expected normal ovarian function and reserve, and the effects of LZ may therefore be different in other patient groups. Wider implications of the findings We confirm previous findings concerning increased follicle growth, increased endogenous FSH and androgen production, and support the rationale for use of LZ co-treatment as a form of endogenous androgen priming sensitizing the follicle to FSH. Letrozole appears to improve the luteal phase with better stimulation of CL and P4 secretion. Trial registration number NCT02939898

Study question Does reducing estradiol levels with letrozole co-treatment during ovarian stimulation with gonadotropins for IVF impact endocrinological and reproductive outcome markers in expected normal responders? Summary answer Letrozole co-treatment maintained follicular phase physiological serum estradiol levels, increased gonadotropin and androgen levels, and increased progesterone in the luteal phase. What is known already Ovarian stimulation for IVF causes supraphysiologic estradiol levels, which exert pituitary suppression reducing gonadotropin stimulation of the corpus luteum. Furthermore, stimulation may increase progesterone in the late follicular phase, reported to impair clinical outcomes, through a putative effect on endometrial maturation and embryo-endometrial asynchrony. Co-treatment with the highly selective aromatase inhibitor letrozole during ovarian stimulation has been shown to reduce estradiol levels and FSH consumption in poor responders, but conflicting data in relation to oocyte yield and implantation rates. The impact of letrozole co-treatment on hormonal changes and reproductive outcome after co-treatment in normal responders remains to be clarified. Study design, size, duration A multicentre double-blinded randomised placebo-controlled trial conducted in 4 fertility clinics at university hospitals in Denmark from August 2016 to November 2018. 159 women were randomised and 129 completed the study; 67 women in the letrozole group and 62 women in the placebo group. The study was conducted in accordance with the Helsinki Declaration and the ICH-Good-Clinical-Practice. Data collection and reporting followed the guidelines of CONSORT to achieve transparent reporting of trials. Participants/materials, setting, methods Women with expected normal ovarian reserve received an antagonist IVF protocol with fixed-dose FSH and fresh single embryo transfer. Co-treatment consisted of once-daily 5 mg letrozole or placebo from the start of stimulation until the day of triggering final oocyte maturation with human chorionic gonadotropin. Serum was collected on 7 visits from stimulation start to 8 days after oocyte retrieval. Clinical pregnancy was determined with a viable foetus by vaginal ultrasound at gestational week 7. Main results and the role of chance The proportion of patients with progesterone >1.5 ng/ml in the late follicular phase was similar in the letrozole versus placebo group with 6% versus 0%, respectively (OR 0, 95 % CI [0;1.6], P =.12). Mid-luteal progesterone levels >30 ng/ml were observed in 59% versus 31%, respectively, of subjects in the letrozole and placebo group (OR 3.3, 95% CI [1.4;7.1], P =.005). Letrozole treatment decreased estradiol levels by 69% (95 % CI [60%;75%], P <.0001) and increased luteinizing hormone (LH), testosterone, and androstenedione levels significantly in both the follicular and luteal phase. Follicle-stimulating hormone (FSH) concentration was elevated in the letrozole group at stimulation day 5 and at trigger day, and overall FSH consumption was diminished. The ongoing pregnancy rate did not differ between the letrozole and placebo group (31% versus 39% (risk-difference of 8%, 95% CI [-25%;11%], P =.55). Letrozole had no significant additional side effects apart from those frequently seen during ovarian stimulation, though a trend towards less nausea and vomiting was observed in the letrozole co-treated group versus the placebo group (28% versus 44% (risk-difference of 16%, 95% CI [-2%;33%], P =.11). Limitations, reasons for caution The diurnal variation of progesterone has been confirmed since this study was completed, hence the timing of the blood samples was not standardized . However, bias is unlikely due to the randomized design. The study was not powered to show an effect on ongoing pregnancy rates. Wider implications of the findings Letrozole co-treatment during ovarian stimulation with gonadotropins maintained serum estradiol at physiological levels, increased follicular phase levels of gonadotropins and androgens, and luteal progesterone levels. These data indicate that letrozole co-treatment may ameliorate the detrimental impacts of gonadotropin stimulation during IVF in normal responders. Trial registration number NCT02939898 and NCT02946684

OBJECTIVE: To evaluate whether diminished ovarian reserve (DOR) and its etiology impact the AMH/AFC ratio?MATERIALS AND METHODS: From November 2018 to December 2021, we conducted a monocentric, retrospective study including a total of 484 infertile patients <37 years with DOR. All patients underwent measurement of AMH levels and AFC. DOR was diagnosed according to the Bologna criteria (AMH<1.1 ng/mL and AFC<7). AMH/AFC ratio was compared to values obtained in 154 tubal or male infertility patients matched for age and BMI, with AMH and AFC in the normal ranges. This ratio was studied according to the etiology of DOR: genetic (n¼26), post-chemotherapy (n¼102), idiopathic (n¼215) or ovarian diseases (ovarian cyst or history of ovarian surgery, n¼141).RESULTS: Overall, median age of women with DOR was 30 (18-37) years. As expected, age and BMI were comparable in women with DOR and those having normal ovarian reserve tests. In addition, the AMH/AFC ratio failed to show any difference between these 2 groups (0.143 AE 0.22 vs. 0.166 AE 0.11, NS, respectively). Among women with DOR, the etiology was significantly associated with different AMH/AFC ratio. Indeed, patient with DOR of surgical origin (ovarian diseases group) displayed higher mean values (0.283 AE 0.32 ng/mL/ Foll) when compared with those included in genetic (0.079 AE 0.15 ng/mL/ Foll, p<0.01), idiopathic (0.103 AE 0.16 ng/ mL/ Foll, p<0.03) or post-chemotherapy (0.084 AE 0.20 ng/mL/ Foll, p<0.01) groups. Moreover, genetic and post-chemotherapy DOR was also associated with lower AMH/AFC ratio in comparison with idiopathic DOR.CONCLUSIONS: AMH/AFC ratio varies according to the etiology of DOR in young women, suggesting different impact on the follicular health, and further oocyte quality.IMPACT STATEMENT: AMH/AFC ratio may represent an innovative tool aiming to indirectly assess follicular health and possibly oocyte quality in young women with DOR. The etiology of DOR differently impacts the follicular function as reflected by AMH/AFC ratio. Further data on live birth rates following natural or medically assisted pregnancies is needed.