Unexplained infertility and age-related infertility: indistinguishable diagnostic entities but different IVF prognosis.

STUDY QUESTION: Is IVF indicated for couples with age-related infertility? SUMMARY ANSWER: IVF may be of doubtful utility for age-related infertility. WHAT IS KNOWN ALREADY: A diagnosis of unexplained infertility is drawn when the diagnostic work-up fails to identify any patent cause. Although typically managed uniformly, unexplained infertility is likely to comprise a wide range of conditions, including age-related infertility (at least in older women). Unfortunately, no validated tests for the identification of age-related infertility exist and these women are typically treated as unexplained cases. However, homologous ART may be less effective for these women because these techniques may be unable to treat the detrimental effects of ageing on oocyte competence. STUDY DESIGN, SIZE, DURATION: Women aged 18-42 years who underwent IVF procedures between January 2014 and December 2021 were selected retrospectively. In the first part of the study, we aimed to assess whether the proportion of women with unexplained infertility (i.e. without patent causes of infertility) increased with age. In the second part of the study, women with unexplained infertility were matched 1:1 by age, study period, and duration of infertility, to those with a patent cause of infertility. If our hypothesis is valid, the first part of the study should highlight an increase in the proportion of unexplained infertility with age. Moreover, in the second part of the study, one should observe a sharper decrease in the rate of IVF success of the procedure with age in women with an unremarkable work-up compared to those with a definite cause of infertility. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women were included if: they had been trying to conceive for more than 2 years, they had retrieved more than three oocytes, and had not undergone previous IVF attempts. We exclude couples with severe male factor (criptozoospermia), chronic anovulation, untreated hydrosalpinx, or intracavitary diseases. The first part of the study aimed at investigating the relative proportion of unexplained infertility with age. The outcome of the second part was the distribution of the live births between unexplained versus explained infertility, in women younger or older than 35 years. Only the results of the first IVF cycle were considered (including both fresh and frozen cycles). The live birth rate corresponded to the cumulative chance of a live birth per oocyte retrieval. MAIN RESULTS AND THE ROLE OF CHANCE: One thousand five hundred and thirty-five women were selected for the first part of the study; 742 of them had unexplained infertility (48%). The frequency of this diagnosis was lower among women aged <35 years (40%) compared to those ≥35 years (52%) (P < 0.001). A clear gradient emerged when considering smaller intervals of age (P < 0.001). A total of 1134 women (567 unexplained cases and 567 explained cases) were selected for the second part of the study. Baseline variables were comparable between women with unexplained and explained infertility. Among women younger than 35 years (n = 229 unexplained cases and 229 explained cases), 108 live births were observed in women with unexplained infertility (47%) and 88 in those with explained infertility (38%). In comparison, among women older than 35 years, the live births occurred in 90 (27%) and 114 (34%) couples, respectively (P = 0.03). The adjusted odds ratio (OR) for a live birth in older women with unexplained infertility was 0.63 (95% CI: 0.43-0.94). In other words, the effectiveness of IVF in older women with unexplained infertility is reduced by an additional 37% when compared to women of similar age with a patent cause of infertility. Moreover, when considering smaller intervals of age, a gradient of the adverse effect of age on the distribution of live births between unexplained and explained infertility emerged (P = 0.003). Overall, these results support the hypothesis that IVF may be of modest benefit in women with age-related infertility. The decline in IVF success is sharper in women with unexplained infertility compared to those with explained infertility, indirectly suggesting that IVF cannot effectively treat age-related infertility. LIMITATIONS, REASONS FOR CAUTION: We postulated that the greater decline in IVF success with age in the unexplained group could be related to the concomitant increase in the proportion of women with age-related infertility. However, even if this is theoretically logical, the unavailability of validated tools to diagnose age-related infertility makes our inference speculative. We cannot exclude that the prevalence of other unknown causes of infertility that cannot also be effectively overcome with IVF could increase with age. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest that IVF may be of modest utility for treating age-related infertility. Offering this procedure to older women with an unremarkable infertility work-up may be questioned. However, the diagnosis of age-related infertility remains challenging and identifying a biomarker that could reliably diagnose age-related infertility is a priority. STUDY FUNDING/COMPETING INTEREST(S): The study was partially funded by the Italian Ministry of Health-current research IRCCS and by a specific grant supported by Ferring. ES declares receiving honoraria for lectures at meetings from IBSA and Gedeon-Richter and he also handles private grants of research from Ferring, IBSA, Theramex, and Gedeon-Richter. All the other authors do not have any conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

The impact of a second embryo biopsy for preimplantation genetic testing for monogenic diseases (PGT-M) with inconclusive results on pregnancy potential: results from a matched case-control study.

PURPOSE: To evaluate whether a second biopsy, following a first diagnostic failure on blastocysts tested for preimplantation genetic testing for monogenic diseases (PGT-M), allows to obtain genetic diagnosis and to what extent this procedure can influence clinical pregnancy and live birth rates compared to the PGT-M process with a successful genetic diagnosis from the first biopsy. METHODS: Embryos from women who underwent PGT-M in an infertility centre and who had been transferred after two biopsies for genetic analysis (n = 27) were matched in a 1:1 ratio accordingly to women's age (± 1 year) and fertility status (fertile vs infertile), as well as with the study period, with embryos who were transferred after receiving a conclusive PGT result straight after the first biopsy (n = 27). The main evaluated outcome was clinical pregnancy rate following embryo transfers in which healthy embryos were transferred after only one biopsy and those in which an embryo was transferred after being re-biopsied. Live birth rate was the secondary outcome. RESULTS: Clinical pregnancy rate was 52% (95% CI: 34-69) following the transfer of a single-biopsy blastocyst and 30% (95% CI: 16-48) following the transfer of a re-biopsied blastocyst. The likelihood to have a healthy baby was 33% (95% CI: 19-52) following the transfer of a blastocyst biopsied once and 22% (95% CI: 11-41) following the transfer of a re-biopsied blastocyst. CONCLUSIONS: The re-biopsy intervention seems to considerably reduce the pregnancy potential of a blastocyst. However, a greater sample size is necessary to clarify this issue definitively.

Return rate following a live birth obtained with ART: frequency and determinants.

PURPOSE: To understand how often couples return to ART centres for a second child. METHODS: Retrospective monocentric cohort study including women who had a first live birth with IVF. The primary objective was to assess the rate of those returning for a second child within five years of the previous pregnancy. The secondary aim was to disentangle the determinants of this rate. RESULTS: A total of 374 patients were included, of whom 188 returned (50%, 95% CI 45-55%). Among those who did not return (n = 186), four (2%) referred to another ART Center and 24 were unreachable. Of the 158 contacted subjects that did not refer for ART, 53 (34%, 95% CI 27-41%) conceived naturally, 57 (36%, 95% CI 29-44%) abandoned their intent of parenthood, and 48 (30%, 95% CI 24-38%) unsuccessfully attempted natural conception. These 48 women (13%) who expressed interest in a second child but did not undergo ART were compared to those seeking a second pregnancy through ART. Baseline characteristics were similar except for an older age (Median 36, IQR: 34-38 vs 34, IQR: 32-36, p = 0.001). Additionally, in terms of IVF cycle characteristics, women who did not return were more likely to achieve their first pregnancy with a fresh transfer rather than a frozen transfer (75% vs 59%, p = 0.05). They also had a higher number of retrieved oocytes (Median 10, IQR: 7-13 vs 9, IQR: 5-12) and less frequently cryopreserved embryos (27% vs 52%, p = 0.003). CONCLUSION: The proportion of couples who have conceived with ART and who are interested in having a second child is high. Our results underline the importance of paying more attention to the number of intended children, as this information could influence clinical management.

Conventional in vitro fertilisation rather than intracytoplasmic sperm injection when only one oocyte is retrieved: Time to overcome irrational fears.

BACKGROUND: The use of intracytoplasmic sperm injection (ICSI) currently extends beyond male factor infertility, notably replacing conventional in vitro fertilisation (IVF) in scenarios like limited oocyte availability, where it is used as a precaution against complete fertilisation failure. While existing studies on the use of conventional IVF in such situations provide some reassurance, the available evidence is somewhat insufficient and ICSI is commonly used. AIMS: To evaluate whether conventional IVF can be a feasible option when only one oocyte is retrieved. MATERIALS AND METHODS: A retrospective study was performed to evaluate the fertilisation rate with conventional IVF in women retrieving only one oocyte and whose partner had normal semen. The study aimed at evaluating whether the fertilisation rate was aligned with the threshold indicated by recognized IVF laboratory performance indicators (Vienna Consensus). Clinical pregnancy and live birth rates were secondary outcomes. RESULTS: Out of 304 cycles with a single oocyte inseminated with conventional IVF, 209 achieved normal fertilisation and 82 did not. Thirteen had no mature oocytes. The fertilisation rate was 69% (95% CI: 63-74%) and increased to 72% (95% CI: 66-77%) when immature oocytes were excluded. The fertilisation rate surpassed the minimum competency threshold of the Vienna Consensus (60%), even if below the benchmark value (75%). Clinical pregnancy and live birth rates per oocyte retrieval were 10% and 8%, respectively. Univariate and multivariate analyses failed to identify any predictive factor of fertilisation. CONCLUSION: Conventional IVF with one oocyte met Vienna Consensus standards even if it fell short of higher benchmarks.

Prevalence and clinical effect of caesarean scar defects in women undergoing IVF.

RESEARCH QUESTION: How common are caesarean scar defects (isthmocele) among patients who have had previous caesarean delivery undergoing IVF for secondary infertility? Does the presence of isthmocele affect the chances of success of IVF? DESIGN: In this cohort study, women referred to an Italian public assisted reproduction centre between January 2016 and April 2021 were retrospectively reviewed. Women with a history of caesarean delivery and an indication for IVF were selected. On the basis of the local policy, all patients with a history of caesarean section underwent saline contrast sonography (SCS). Sonographic evaluation was standardized. RESULTS: One hundred and forty-four women were eligible, of whom 22 declined SCS and eight decided to delay pregnancy seeking. Overall, 114 women were available for data analysis. Seventy-six women were diagnosed with caesarean scar defects, corresponding to a prevalence of 67% (95% CI 58 to 75%). Baseline characteristics of women with and without isthmocele were similar. Conversely, the clinical pregnancy rate (adjusted OR 0.31, 95% CI 0.13 to 0.72) and live birth rate (adjusted OR 038, 95% CI 0.17 to 0.86) were significantly lower among affected women. No associations between specific sonographic defect characteristics and IVF outcome could be identified. CONCLUSIONS: Caesarean scar defects are common among women with a history of caesarean section requiring IVF. The presence of these lesions may reduce the chance of success of the procedure.

Adherence to the Mediterranean diet and the risk of unexpected poor response to ovarian stimulation in IVF cycles.

RESEARCH QUESTION: Can preconception adherence to a Mediterranean diet influence the rate of poor response to ovarian stimulation in IVF cycles? DESIGN: The impact of dietary habits on the success of IVF is controversial. Inconsistencies may be explained by confounders associated with the use of pregnancy as an outcome as well as by a reductionist view of diet that focuses on single components rather than on food patterns. This cross-sectional study analysed adherence to a Mediterranean diet in women with unexpected poor response to ovarian stimulation. Main inclusion criteria were: age 18-39 years, normal weight, preserved ovarian reserve and starting dose of gonadotrophins of 150-225 IU/day. Adherence to the Mediterranean diet was assessed through a Mediterranean diet score (MDS). Unexpected poor ovarian response was defined as the retrieval of ≤3 suitable oocytes. RESULTS: A total of 296 women were included, of whom 47 (15.9%) showed an unexpected poor response. A clear dose-related association with tertiles of MDS was not observed in the univariate analysis. However, in the multivariate analysis, the risk of unexpected poor response was significantly lower for women in the second tertile of MDS compared with the first tertile (adjusted odds ratio [OR] 0.29, 95% confidence interval [CI] 0.11-0.76) and for women in the second and third tertiles, grouped together, compared with the first tertile (adjusted OR 0.34, 95% CI 0.14-0.82). CONCLUSIONS: Low adherence to a Mediterranean diet could be a risk factor for unexpected poor ovarian response.

Is endometrial receptivity affected in women with endometriosis? Results from a matched pair case-control study of assisted reproductive technology treatments.

RESEARCH QUESTION: Is endometriosis detrimental to embryo implantation? DESIGN: A retrospective matched case-control study of women with a surgical or ultrasound diagnosis of endometriosis at Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico di Milano between 2015 and 2021. Women with endometriosis who underwent a 'freeze-all' cycle during an IVF treatment were eligible to be included. They were matched to patients without the disease, who also underwent cryopreserved blastocyst transfer cycles, in a 1:1 ratio by age (±1 year), and number (=) and quality (±1 top versus low) of cryopreserved blastocysts. All women underwent single frozen embryo transfer, and assisted reproductive technology outcomes suggested by the Core Outcome Measure for Infertility Trials initiative were evaluated. The main outcome was the cumulative live birth rate per cycle. RESULTS: One hundred and one women with endometriosis and 101 matched unaffected women were included. Cumulative live birth rate per cycle did not vary between women with and without endometriosis (50% versus 58%, respectively; P = 0.32). On the basis of the Kaplan-Meier analysis, the predicted success rates over four embryos transferred were also similar (74% versus 82%, respectively; P = 0.67). CONCLUSION: In women with moderate or severe endometriosis, these retrospective results seem to indicate no or a limited effect of the disease on endometrial receptivity.

Conventional IVF performs similarly in women with and without endometriosis.

PURPOSE: A reduced oocyte competence has been claimed as one of the factors underlying infertility in women with endometriosis. This idea has justified the hypothesis that intracytoplasmic sperm injection (ICSI), rather than conventional IVF (c-IVF), may overcome oocyte impairment and ensure better assisted reproduction technology (ART) outcomes; however, data from the literature are controversial. Thus, the aim of this study was to compare ART success rates following (c-IVF) between women with and without endometriosis in the presence of normozoospermic partners. METHODS: This is a retrospective, matched case-control study of 314 patients who underwent c-IVF cycles between January 2014 and December 2020. Women with endometriosis were matched in a 1:1 ratio with patients undergoing ART for other indications, considering age (± 6 months), number of oocytes retrieved (± 1), and study period. The main outcome measures included total fertilization failure, fertilization rate, embryo quality, cumulative clinical pregnancy, and live birth rates. RESULTS: The fertilization rate and the proportion of women with total fertilization failure did not differ between women with and without endometriosis. Similarly, all other embryological variables did not also differ, except for the number of top-quality cleavage stage embryos which was higher in the endometriosis group. Cumulative clinical pregnancy and live birth rates were similar between women with and without endometriosis. CONCLUSION: A diagnosis of endometriosis does not negatively affect the performance of c-IVF; thus, c-IVF can be efficiently used in women affected, unless a male factor is also involved. This issue holds clinical relevance to help operators on their insemination technique decision-making.

Follicular steroidogenesis in random start protocols for oocyte cryopreservation.

PURPOSE: Random start protocols are commonly used for oocyte cryopreservation in women with cancer. However, albeit generally reassuring, available evidence is still insufficient to rule out a sub-optimal cycle outcome. This study aimed to compare follicular steroidogenesis between women initiating the random start protocol in the luteal phase and those initiating in the follicular phase. METHODS: Consecutive women with cancer scheduled for oocyte cryostorage were prospectively recruited. We excluded those requiring a concomitant letrozole assumption. All women received a standardized protocol with recombinant FSH and GnRH antagonists. At the time of oocyte retrieval, follicular fluids were pooled, and a sample was collected and frozen at -80 °C. All samples were assayed concomitantly after thawing by liquid chromatography-tandem mass spectrometry. The concentration of 15 different steroid hormones was determined. RESULTS: Seventy-one women were recruited. Thirty-three initiated the ovarian stimulation in the luteal phase, while the remaining 38 initiated in the follicular phase. Baseline characteristics were generally similar. Cycle outcome did also not differ; the median (interquartile range) number of frozen mature oocytes was 9 (5-14) and 10 (5-21), respectively (p = 0.42). None of the 15 tested steroid hormones differed. CONCLUSIONS: The endocrine microenvironment surrounding oocytes is not markedly influenced by the phase of the menstrual cycle at the initiation of ovarian stimulation. This result further supports the validity of random start protocols.

The impact of endometriosis on IVF efficacy: qualitative and quantitative assessment of ovarian response and embryo development.

RESEARCH QUESTION: The detrimental effects of endometriosis on IVF success are well known, but the underlying mechanisms are still uncertain. The objective of this study was to investigate the possible detrimental effects of the disease on ovarian response and embryo development. DESIGN: IVF cycles performed in the authors' unit between 2014 and 2020 were retrospectively reviewed. Women with a good ovarian reserve who underwent their first IVF cycle were included. A total of 248 women with endometriosis were identified and matched to 248 women without the disease, according to age, type of stimulation, anti-Müllerian hormone concentration and study period. The primary outcome was the number of women without good-quality embryos. Secondary outcomes included the rate of unexpected poor response (retrieval of ≤3 oocytes), the cumulative clinical pregnancy rate and the cumulative live birth rate. RESULTS: The number of women without good-quality embryos did not differ between women with endometriosis (exposed group) and those without it (unexposed group). The adjusted odds ratio was 0.85 (95% CI 0.51-1.44, P = 0.56). The clinical pregnancy rate and the live birth rate were also similar. Conversely, the rate of unexpected poor response was higher among women with endometriosis (23% versus 13%, P = 0.005). CONCLUSIONS: Overall, endometriosis does not markedly affect folliculogenesis and embryo development in the context of IVF. Only an increased risk of unexpected poor response emerged.

Endometrial preparation does not affect the risk of hypertensive disorders of pregnancy in low-risk women undergoing frozen embryo transfer.

OBJECTIVE: Frozen embryo transfer (FET) is associated with a higher risk of hypertensive disorders in pregnancy. The objective of the present study is to evaluate the effect of different protocols of endometrial preparation on the risk of these disorders. METHODS: We conducted a retrospective cohort study on 594 singleton pregnancies achieved by embryo transfer of single frozen-thawed blastocysts. Women with preexisting risk factors for hypertensive disorders were excluded. Women were divided into two groups according to the endometrial preparation protocol: either natural cycle (n = 495) or programming cycle with hormonal replacement therapy (n = 97). The primary outcome was the frequency of hypertensive disorders in pregnancy: specifically, gestational hypertension and preeclampsia. RESULTS: No differences emerged between women following the natural cycle and those following the programming cycle in the frequency of gestational hypertension (5 vs. 4%) and preeclampsia (1.1 vs. 1.2%). No impact emerged also after multivariate analyses. CONCLUSIONS: Women receiving hormonal replacement therapy have the same risk of gestational hypertension and preeclampsia as women following natural cycles when considering low-risk singleton pregnancies.

Effect of letrozole on follicular fluid steroids concentrations in cancer patients undergoing oocyte cryopreservation.

PURPOSE: To investigate the impact of letrozole administration on follicular steroid hormones during controlled ovarian hyperstimulation for fertility preservation. METHODS: One hundred and nineteen women with cancer undergoing oocytes retrieval for fertility preservation were recruited. All women underwent ovarian hyperstimulation according to a random start protocol. Those with hormone-sensitive tumors also received letrozole, an aromatase inhibitor aimed at keeping peripheral estrogen levels low. At the time of oocytes retrieval, a sample of follicular fluid was collected and frozen. All samples were assayed concomitantly after thawing, by liquid chromatography tandem mass spectrometry. The concentration of 15 steroid hormones was determined and results were compared between women who did and did not receive letrozole. RESULTS: Fifty-two women were treated with letrozole, while 67 were not. Statistically significant differences emerged for 12 of the 15 tested steroids. They were the following: cortisol, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone sulfate (DHEAS), dehydroepiandrosterone (DHEA), estradiol, androstenedione, testosterone, dihydrotestosterone (DHT), 17-hydroxyprogesterone, progesterone and corticosterone. The most striking differences were observed for testosterone that showed a more than 200-time increase in women receiving letrozole. Estradiol was conversely reduced to a third. CONCLUSIONS: The endocrine microenvironment surrounding oocytes is markedly perturbed by the concomitant assumption of letrozole. Robust clinical evaluation is pressingly needed to rule out any detrimental effect on the chance of live birth with the use of these oocytes.

Monozygotic twin rate among ART centers: a multicenter analysis of data from 18 Italian units.

PURPOSE: The risk of monozygotic twins (MZTs) is increased in couples undergoing assisted reproductive technology (ART) treatments. Several systematic reviews have investigated the possible determinants linked to ART, but results obtained have not been conclusive. The study aims to investigate whether the incidence of MZT differed among ART centers. METHODS: This is a multicenter retrospective cohort study using the Italian ART National Registry database and involving the centers reporting data from individual ART cycles from 2015 to 2019. To investigate the incidence of MZT, only single embryo transfer cycles were considered. Women who had sex-discordant deliveries were excluded. MZT rate was calculated as the number of multiple pregnancies (more than one gestational sac at first ultrasound) out of the total number of clinical pregnancies. A binomial distribution model was used to determine the 95% CI of the frequency of MZT. RESULTS: Eighteen centers were included, and they provided data on 10,433 pregnancies. The total number of MZT was 162, corresponding to an incidence of 1.5% (95% CI: 1.3-1.8%). The rate of MZT among centers varied between 0% (95% CI: 0.0-25.9%) and 3.2% (95% CI: 1.3-8.1%). All the 95% CIs included 1.5%, rejecting the hypothesis that the MZT rate may significantly differ among centers. CONCLUSIONS: The rate of MZT did not significantly vary among ART centers. Local factors are unlikely to explain the increased rate of MZT in ART pregnancies.

Oral Vitamin D supplementation impacts gene expression in granulosa cells in women undergoing IVF.

STUDY QUESTION: Does oral Vitamin D supplementation alter the hormonal milieu of follicular fluid (FF) and the transcriptomic profile of luteinised granulosa cells (GCs) in women with Vitamin D deficiency undergoing IVF? SUMMARY ANSWER: A transcriptomic signature relevant to oral Vitamin D supplementation in luteinised GCs was demonstrated, although Vitamin D supplementation did not alter hormone levels in FF. WHAT IS KNOWN ALREADY: Vitamin D deficiency is linked to lower live birth rates among women undergoing IVF. It is unclear whether Vitamin D elicits a targeted action in reproductive physiology or is a surrogate marker of overall well-being. Several in-vitro studies, but none in vivo, have examined the impact of Vitamin D on the periovulatory follicle, focusing on GCs as a proxy marker of oocyte competence. STUDY DESIGN, SIZE, DURATION: We present a report of secondary outcomes from the SUNDRO clinical trial, which was launched in 2016 to determine whether Vitamin D supplementation can improve the IVF outcomes of women who are deficient in Vitamin D (<30 ng/ml). FF samples of 145 women who were randomised to receive Vitamin D or placebo from March 2017 to January 2019 were collected. All follicles that were aspirated in our study measured ≥11 mm on the day of hCG trigger. The first cohort of samples was collected from the dominant follicle of each participant and utilised for hormone profiling (n = 50 Vitamin D, n = 45 Placebo). For the second cohort, the follicle aspirates of each participant were pooled to create a single FF sample, which was used for the isolation of GCs for gene expression studies (n = 20 Vitamin D, n = 30 placebo). Six of the samples from the second cohort were used for RNA-sequencing analysis (n = 3 Vitamin D, n = 3 placebo). PARTICIPANTS/MATERIALS, SETTING, METHODS: Two academic infertility units were involved in the recruitment of the participants, who received a single dose of oral 25-hydroxyvitamin D (600 000 IU) or placebo, 2-12 weeks before oocyte retrieval. Women in both groups were deficient in Vitamin D, aged 18-39 years with a normal BMI (18-25 kg/m2) and <3 previous IVF cycles. The FF was aspirated at the time of oocyte retrieval and stored. Liquid chromatography tandem mass spectrometry was used to measure FF abundance of 25-hydroxyvitamin D, aldosterone, androstenedione, cortisol, cortisone, corticosterone, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone, oestradiol (E2), 17-OH-hydroxyprogesterone, progesterone (P4) and testosterone. GCs were isolated from pooled FFs and the transcriptome was evaluated by RNA-sequencing and RT-PCR. Ingenuity pathway analysis (IPA) was used to assess the top canonical pathways and upstream regulators mediating the action of Vitamin D. MAIN RESULTS AND THE ROLE OF CHANCE: At oocyte retrieval, FF concentration of 25-hydroxyvitamin D was 2.8-fold higher (P < 0.001) in the Vitamin D group (39.5 ng/ml; n = 50) compared to placebo (13.8 ng/ml; n = 45) but no other hormonal differences were detected. In the placebo group, but not the Vitamin D group, weak correlations of 25-hydroxyvitamin D concentration with P4 (r = 0.31, P = 0.03) and E2 (r = 0.45, P = 0.002) were observed. RNA-sequencing identified 44 differentially expressed genes in the GCs of patients who received Vitamin D (n = 3) compared to placebo (n = 3). RT-PCR demonstrated upregulation of VDR (vitamin D receptor), GSTA3 (glutathione S-transferase A3) and IL21R (interleukin 21 receptor), and downregulation of P T GS2 (prostaglandin-endoperoxide synthase 2), KLF4 (kruppel-like factor 4), T RP C4 (transient receptor potential cation channel subfamily C member 4), VEGF (vascular endothelial growth factor), RXRB (retinoid X receptor beta) and AGER (advanced glycosylation end-product specific receptor) genes in the Vitamin D (n = 17) versus placebo (n = 27) group. IPA suggested roles of Vitamin D in antioxidant defence. LIMITATIONS, REASONS FOR CAUTION: Interpretation of the data is influenced by our intervention strategy (2-12 weeks prior to retrieval). As folliculogenesis may last 5-6 months, our protocol can only examine with confidence the impact of Vitamin D on the final stages of follicular growth. Furthermore, we examined the hormonal profile of the dominant follicle only, while the GC data reflect the transcriptome of all (pooled) follicles large enough to be used for IVF. Luteinised GCs from controlled ovarian stimulation were used in this study, which may be functionally distinct from the GCs of developing follicles. Moreover, the sample size for RNA-sequencing analysis was low (n = 3 per group), regardless of validation by RT-PCR that was performed on a larger cohort, introducing complexity to the IPA analysis, which required an input of data with P-adjusted <0.08 instead of <0.05 to be informative. WIDER IMPLICATIONS OF THE FINDINGS: This is the first in-vivo study to show that Vitamin D supplementation alters gene expression in luteinised GCs. In contrast to some in-vitro evidence, no effect of the intervention on expression of genes encoding steroidogenic enzymes was observed. Unlike other studies, our results suggest that supplementation with Vitamin D is unlikely to directly influence hormone availability in FF. Our findings instead reinforce the hypothesis that Vitamin D could be considered one of the gatekeepers in protecting against an exaggerated response to ovarian stimulation. STUDY FUNDING/COMPETING INTEREST(S): The study has been funded by the Italian Ministry of Health (RF-2013-02358757) following peer review in the competitive 'Bando di Ricerca Finalizzata e Giovani Ricercatori 2013' for the clinical trial SUNDRO (EudraCT registration number 2015-004233-27). There are no competing interests. TRIAL REGISTRATION NUMBER: EudraCT registration number 2015-004233-27.

Should rescue ICSI be re-evaluated considering the deferred transfer of cryopreserved embryos in in-vitro fertilization cycles? A systematic review and meta-analysis.

BACKGROUND: Total fertilization failure represents a particularly frustrating condition for couples undergoing in vitro fertilization. With the aim of reducing the occurrence of total fertilization failure, intracytoplasmic sperm injection (ICSI) has become the first choice over conventional in vitro fertilization (IVF) procedures although evidence of improved results is still debated and its use in couples without male factor infertility is not recommended. Among the strategies potentially useful to promote the use of conventional IVF, we herein call attention to the late rescue ICSI, which consists in performing ICSI after 18-24 h from conventional insemination on oocytes that show no signs of fertilization. This treatment has however been reported to be associated with a low success rate until recent observations that embryos derived from late rescue ICSI may be transferred after cryopreservation in a frozen-thawed cycle with improved results. The aim of the present study was to assess whether frozen embryos deriving from rescue ICSI performed about 24 h after conventional IVF may represent a valuable option for couples experiencing fertilization failure. METHODS: A systematic review on the efficacy of late rescue ICSI was performed consulting PUBMED and EMBASE. RESULTS: Including twenty-two original studies, we showed that clinical pregnancy rate per embryo transfer and implantation rate obtainable with fresh embryo transfers after rescue ICSI are not satisfactory being equal to 10 and 5%, respectively. The transfer of cryopreserved rescue ICSI embryos seems to offer a substantial improvement of success rates, with pregnancy rate per embryo transfer and implantation rate equal to 36 and 18%, respectively. Coupling rescue ICSI with frozen embryo transfer may ameliorate the clinical pregnancy rate for embryo transfer with an Odds Ratio = 4.7 (95% CI:2.6-8.6). CONCLUSION: Results of the present review support the idea that r-ICSI coupled with frozen embryo transfer may overcome most of the technical and biological issues associated with fresh transfer after late r-ICSI, thus possibly representing an efficient procedure for couples experiencing fertilization failure following conventional IVF cycles. TRIAL REGISTRATION: Prospero registration ID: CRD42021239026 .

Fibroids and natural fertility: a systematic review and meta-analysis.

Available evidence from IVF studies supports a detrimental effect of submucosal and intramural fibroids on embryo implantation. It is misleading, however, to infer evidence obtained in IVF settings to natural fertility. Therefore, a systematic review and meta-analysis was conducted on the effect of fibroids on natural fertility. Studies comparing fertile and infertile women, and those investigating whether the presence of fibroids was a risk factor, were reviewed, as well as studies comparing women with and without fibroids. The aim was also to establish whether the frequency of infertility differed between the two groups. Seven out of 11 selected studies did not aim to establish whether fibroids caused infertility but, rather, whether a history of infertility could be a risk factor for fibroids. A meta-analysis of the four remaining studies that concomitantly evaluated the presence of fibroids and infertility studies highlighted a common odds ratio of fibroids in subfertile women of 3.54 (95% CI 1.55 to 8.11). When focusing on the two most informative studies, i.e. the studies comparing time to pregnancy in women with and without fibroids, the common OR was 1.93 (95% CI 0.89 to 4.18). In conclusion, the association between fibroids and infertility has been insufficiently investigated. Epidemiological studies suggest, but do not demonstrate, that fibroids may interfere with natural fertility. Given the high prevalence of these lesions in women seeking pregnancy, further evidence is urgently needed.

Single oral dose of vitamin D3 supplementation prior to in vitro fertilization and embryo transfer in normal weight women: the SUNDRO randomized controlled trial.

BACKGROUND: Improving in vitro fertilization success is an unmet need. Observational studies have suggested that women with deficient or insufficient vitamin D have lower chances of in vitro fertilization success, but whether supplementation improves clinical pregnancy rate remains unclear. OBJECTIVE: This study aimed to determine whether oral vitamin D3 supplementation improves clinical pregnancy in women undergoing an in vitro fertilization cycle. STUDY DESIGN: The "supplementation of vitamin D and reproductive outcome" trial is a 2-center randomized superiority double-blind placebo-controlled trial. Subjects were recruited between October 2016 and January 2019. Participants were women aged 18 to 39 years with low vitamin D (peripheral 25-hydroxyvitamin D of <30 ng/mL), serum calcium of ≥10.6 mg/dL, body mass index of 18 to 25 kg/m2, and antimüllerian hormone levels of >0.5 ng/mL and starting their first, second, or third treatment cycle of conventional in vitro fertilization or intracytoplasmic sperm injection. The primary outcome was the cumulative clinical pregnancy rate per cycle. Pregnancies obtained with both fresh or frozen embryo transfers were included. Clinical pregnancy was defined as an intrauterine gestational sac with a viable fetus. The primary analysis was performed according to the intention-to-treat principle and could also include natural conceptions. Secondary outcomes included total dose of gonadotropins used, embryologic variables (number of oocytes retrieved, number of suitable oocytes retrieved, fertilization rate, and rate of top-quality embryos), and clinical outcomes (miscarriage rate and live birth rate). RESULTS: Overall, 630 women were randomized 2 to 12 weeks before the initiation of the in vitro fertilization cycle to receive either a single dose of 600,000 IU of vitamin D3 (n=308) or placebo (n=322). Interestingly, 113 (37%) and 130 (40%) women achieved a clinical pregnancy in the treatment and placebo groups, respectively (P=.37). The risk ratio of clinical pregnancy in women receiving vitamin D3 was 0.91 (95% confidence interval, 0.75-1.11). Compared with the placebo, vitamin D3 supplementation did not improve the rate of clinical pregnancy. Exploratory subgroup analyses for body mass index, age, indication to in vitro fertilization, ovarian reserve, interval between drug administration and initiation of the cycle, and basal levels of 25-hydroxyvitamin D failed to highlight any clinical situation that could benefit from the supplementation. CONCLUSION: In women with normal weight with preserved ovarian reserve and low vitamin D levels undergoing in vitro fertilization cycles, a single oral dose of 600,000 IU of vitamin D3 did not improve the rate of clinical pregnancy. Although the findings do not support the use of vitamin D3 supplementation to improve in vitro fertilization success rates, further studies are required to rule out milder but potentially interesting benefits and explore the effectiveness of alternative modalities of supplementation.

How common is real repeated implantation failure? An indirect estimate of the prevalence.

RESEARCH QUESTION: What is the real prevalence of repeated implantation failure (RIF) and what reliable estimates can be given on the risk of false-positive diagnosis after two or three failed IVF attempts. DESIGN: A recent theoretical model suggested that commonly used definitions (two or three failed IVF attempts in good-prognosis couples) may expose couples to substantial odds of overdiagnosis and overtreatment. This model, however, was theoretical and based on unproven assumptions that the pregnancy rate in the non-RIF population was 30% and the prevalence of RIF was 10%. In the present study, we applied this model to real data to distinguish the real prevalence of RIF and to provide more reliable estimates on the risk of false-positive diagnosis after two or three failed IVF attempts. To this aim, we retrospectively selected 1221 good-prognosis couples and evaluated pregnancy rates up to the third cycle. RESULTS: The clinical pregnancy rate at first, second and third IVF cycle was 52%, 41% and 28%, respectively. A pregnancy rate of 61% was extrapolated in the non-RIF population and 15% among women who had experienced RIF. Therefore, the rate of false-positive diagnoses of RIF after two, three and six failed cycles would be 46%, 25%, and 2%, respectively. CONCLUSIONS: Our analyses show that estimated prevalence of RIF is 15%. The frequently used definition of RIF based on three failed attempts (but not two) in good-prognosis couples seems justified. Physicians, however, should bear in mind that couples may be inappropriately labelled with this condition in one out of four cases.

The role of diet in unexpected poor response to ovarian stimulation: a cross-sectional study.

RESEARCH QUESTION: Is there an association between diet and poor ovarian response to ovarian stimulation in women with normal biomarkers of ovarian reserve? DESIGN: Women eligible for IVF at an Academic Fertility Center were invited to participate in this prospective cross-sectional study. The main inclusion criteria were age 18-39 years, body mass index 18-25 kg/m2, preserved ovarian reserve (antral follicle count 10-22 or anti-Müllerian hormone concentration 2-5 ng/ml) and a starting dose of gonadotrophins of 150-225 IU/day. Information on diet was obtained using a validated food frequency questionnaire. 'Unexpected poor ovarian response' was defined as the retrieval of three or fewer suitable oocytes. A logistic regression model was used to adjust for confounders. RESULTS: Out of the 303 women enrolled in the study, 48 (16%) showed an unexpected poor ovarian response. The frequency of poor responders increased with increasing glycaemic load, carbohydrate intake and fibre intake. When comparing the third with the first tertile (reference), the adjusted odds ratios for these were 3.91 (95% confidence interval [CI] 1.11-13.83, P = 0.04), 4.78 (95% CI 1.23-18.51, P = 0.02) and 6.03 (95% CI 1.18-30.77, P = 0.07), respectively. CONCLUSIONS: Elevated dietary glycaemic load as well as carbohydrate intake and fibre intake is significantly associated with unexpected poor ovarian response. Future interventional studies should clarify whether dietary modification might restore normal response.

Effect of vitamin D supplementation on assisted reproduction technology (ART) outcomes and underlying biological mechanisms: protocol of a randomized clinical controlled trial. The "supplementation of vitamin D and reproductive outcome" (SUNDRO) study.

BACKGROUND: Vitamin D plays an important role in human physiology and pathology. The receptor for vitamin D regulates 0.5-5% of the human genome. Accordingly, vitamin D insufficiency has been shown to increase the risk of several diseases. In recent years, based on growing evidence, on a role of vitamin D has been also postulated in reproductive health both in animals and humans, especially in female fertility female fertility. In vitro fertilization success was shown to be higher in women with appropriate reserves of vitamin D. However a causal relation has not been demonstrated and randomized controlled trials testing the effectiveness of vitamin D supplementation in IVF are warranted. METHODS: This is a multicenter randomized double blinded placebo controlled study aimed at determining the benefits of vitamin D [25(OH)D] supplementation in improving clinical pregnancy rate in women undergoing IVF. Eligible women with a serum level of 25-hydroxyvitamin D [25(OH)D] < 30 ng/ml will be randomized. Recruited women will be given the drug (either 600,000 IU of 25(OH) D or placebo in a single oral administration) at the time of randomization. Two centres will participate and the sample size (700 women) is foreseen to be equally distributed between the two. Patients will be treated according to standard IVF protocols. DISCUSSION: The primary aim of the study is the cumulative clinical pregnancy rate per oocyte retrieval. Clinical pregnancy is defined as the presence of at least one intrauterine gestational sac with viable foetus at first ultrasound assessment (3 weeks after a positive human chorionic gonadotropin [hCG] assessment). Secondary outcomes include: 1) clinical and embryological variables; 2) oocyte and endometrium quality at a molecular level. To investigate this latter aspect, samples of cumulus cells, follicular and endometrial fluids will be obtained from a subgroup of 50 age-matched good-prognosis cases and controls. TRIAL REGISTRATION: The protocol was included in EudraCT on 22nd September 2015 with the registration number assigned ' 2015-004233-27 '; it was submitted through the database of the Italian "Osservatorio Nazionale della Sperimentazione Clinica (OsSC)" - (National Monitoring Centre of Clinical Trials) to the National Competent Authority on 8th March 2016 and approved on 23rd June 2016.