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(1) Background: The aim of our study is to evaluate whether cell-free DNA testing can overlap the genetic testing of miscarriage tissue in women with early pregnancy loss (EPL) and length of recurrent pregnancy loss (RPL); (2) Methods: We conducted a prospective cohort study at the Pregnancy Loss Unit of the Fondazione Policlinico Universitario A. Gemelli (IRCCS), Rome, Italy between May 2021 and March 2022. We included women with EPL and length of RPL. Gestational age was >9 weeks + 2 days and 25 and

STUDY QUESTION Do low levels of anti-Müllerian hormone (AMH) or antral follicle count (AFC) properly predict miscarriage in young women conceiving with ART? SUMMARY ANSWER Low ovarian reserve, as indicated by AMH or AFC, is not associated with miscarriage in young women conceiving with ART. WHAT IS KNOWN ALREADY Presently, the impact of low ovarian reserve on the risk of miscarriage remains controversial. Some studies have reported an association between serum AMH levels and AFC and miscarriage, but others have failed to confirm these findings. The main limitation that undermines the reliability and consistency of the results is the confounding effect of female age. Indeed, after 35 years of age, on the one hand, the risk of miscarriage starts increasing because of impaired oocyte quality while, on the other, the physiological decline in AMH and AFC levels continues, thus hampering the possibility to properly explore the real effects of reduced ovarian reserve. Indeed, the two processes, i.e., the gradual loss of resting primordial follicles and the loss of oocyte quality, progress in parallel. In other words, the older the woman becomes, the higher is the risk of miscarriage, but one cannot distinguish between the effects of biological aging on oocyte quality and those mediated by a lower ovarian reserve. STUDY DESIGN, SIZE, DURATION The present retrospective monocentric cohort study was carried out at Fondazione IRCSS Ca Granda Ospedale Maggiore Policlinico, Milan. All women referred to the ART Unit between 2014 and 2021 and who underwent either conventional in vitro fertilization (c-IVF), intracytoplasmatic sperm injection (ICSI) or intrauterine insemination (IUI) were reviewed. Only women younger than 35 were eligible because, up to this age, the risk of miscarriage is steady and not strictly related to age. PARTICIPANTS/MATERIALS, SETTING, METHODS Women younger than 35 who achieved a singleton clinical pregnancy with c-IVF, ICSI or IUI were selected. Women with patent causes of recurrent miscarriage were excluded, as well as those undergoing pregnancy termination for fetal or medical causes. Women who did and did not have a pregnancy loss before 20 weeks’ gestation were compared. Detailed information was obtained from charts of the consulting patients. ART procedures were performed according to the standardized policy of our Unit. All women underwent serum AMH measurement and a transvaginal assessment of AFC prior to initiation of treatment. AMH levels were measured by a commercially available ELISA assay. To assess AFC, all identifiable antral follicles 2–10 mm in diameter at ultrasound were recorded. The primary outcome was the risk of miscarriage for women with serum AMH levels below 5 pmol/l. MAIN RESULTS AND THE ROLE OF CHANCE There were 538 women were included, of whom 92 (17%) had a miscarriage. The areas under the ROC curves for prediction of miscarriage based on AMH levels and AFC were 0.51 (95%CI: 0.45-0.58) and 0.52 (95%CI: 0.45-0.59), respectively. The OR of miscarriage for women with serum AMH levels below 5.0 pmol/l was 1.10 (95%CI: 0.51-2.36); the adjusted OR was 1.12 95%CI: 0.51-2.45). Analyses were repeated considering other thresholds for AMH (2.9, 3.6 and 7.9 pmol/l) and for AFC (thresholds of 7 and 10). No associations emerged. LIMITATIONS, REASONS FOR CAUTION The retrospective design of the study hampered the collection of more precise but potentially relevant clinical information of the couples. We did not exclude women suffering from PCOS, a condition possibly associated with miscarriage. Moreover, the baseline characteristics of women who did and did not have a miscarriage differed in some characteristics. Thus, we adjusted the OR using a multivariate analysis, but we cannot fully exclude residual confounding effects. Finally, our results cannot be inferred to women older than 35. The mechanisms causing premature exhaustion of ovarian reserve may be different in younger and older women and this may lead to a different impact on the risk of miscarriage. WIDER IMPLICATIONS OF THE FINDINGS Women embarking on ART with low ovarian reserve should be informed of their likely poor response to ovarian stimulation but can be reassured that, if conception occurs, their risk of miscarriage is not increased. STUDY FUNDING/COMPETING INTEREST(S) This study was partially funded by Italian Ministry of Health - Current research IRCCS. Prof. Somigliana reports grants from Ferring and honoraria for lectures from Merck-Serono and Gedeon-Richter. All the other authors do not have any competing interest to declare. TRIAL REGISTRATION NUMBER N/A WHAT DOES THIS MEAN FOR PATIENTS? The impact of low ovarian reserve on the risk of miscarriage remains controversial. Serum anti-Müllerian hormone (AMH) and ultrasound assessment of antral follicle count (AFC) are both biomarkers commonly used to assess ovarian reserve. Some studies have reported an association between AMH and/or AFC and miscarriage, but others failed to confirm these findings. In this regard, female age represents the most important limitation for the reliability and consistency of the results. Indeed, after 35 years of age, the risk of miscarriage starts increasing because of impaired oocyte quality but, at the same time, there is a physiological decline in AMH and AFC levels. In other words, the older the woman, the higher the risk of miscarriage. In ART, however, the effects of biological aging on oocyte quality and those mediated by a lower ovarian reserve cannot be distinguished during assisted reproduction technology (ART) treatments. To address this issue, we retrospectively selected young women who had a clinical pregnancy through ART and evaluated whether low levels of AMH or AFC could predict miscarriage. All women under 35 who were referred to our ART Unit and who underwent ART procedures were recruitable. This study included 538 women, of whom 92 (17%) had a miscarriage. No associations emerged in terms of AMH levels and AFC between women who did and did not have a pregnancy loss before 20 weeks’ gestation. Despite some limitations, we can conclude that serum AMH levels are not associated with miscarriage in young women conceiving with ART. Young women embarking on ART with low ovarian reserve should therefore be informed of their poorer response to ovarian stimulation but can be reassured that, if conception occurs, their risk of miscarriage is not increased.

PURPOSE: To assess whether live birth rates (LBR) and maternal/neonatal complications differed following single fresh and frozen-warmed blastocyst transfer. METHODS: The present retrospective observational study analyzed 4,613 single embryo transfers (SET) (646 fresh and 3,967 frozen) from January 1, 2014, to December 31, 2018. Fresh embryo transfer at blastocyst stage was considered according to the age of the patient and her prognosis. In case of the risk of ovarian hyperstimulation syndrome, premature progesterone rise, non-optimal endometrial growth, or supernumerary embryos, cryopreservation with subsequent frozen embryo transfer (FET) was indicated. RESULTS: No differences in LBR were recorded. Fresh embryo transfers yielded an increase both in neonatal complications OR 2.15 (95% CI 1.20–3.86, p 0.010), with a higher prevalence of singletons weighting below the 5th percentile (p 0.013) and of intrauterine growth retardation (p 0.015), as well as maternal complications, with a higher placenta previa occurrence OR 3.58 (95% CI 1.54–8.28, p 0.003), compared to FET. CONCLUSION: LBR appears not to be affected by the transfer procedure preferred. Fresh embryo transfer is associated with higher risk of neonatal complications (specifically a higher prevalence of singletons weighting below the 5th percentile and of intrauterine growth retardation) and placenta previa. Reflecting on the increased practice of ART procedures, it is imperative to understand whether a transfer procedure yields less complications than the other and if it is time to switch to a “freeze-all” procedure as standard practice. Trial registration: Clinical Trial Registration Number: NCT04310761. Date of registration: March 17, 2020, retrospectively registered. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02362-3.

BACKGROUND Anti-Müllerian hormone (AMH) serum concentration and antral follicle count (AFC), as measured by transvaginal ultrasonography, accurately reflect the antral follicle pool. However, AMH and AFC association with fertility surrogates (i.e. age at menopause, probability of conceiving naturally and ART success rate) is questioned. Miscarriage is often considered an alternative measure of reproductive capacity. Nonetheless, the impact of diminished ovarian reserve (DOR) on miscarriage incidence remains an understudied and unresolved issue. OBJECTIVE AND RATIONALE The aim of this systematic review and meta-analysis was to elucidate associations between DOR and miscarriage risk, both in women who conceived naturally and in those who achieved pregnancy through ART. SEARCH METHODS Relevant studies were identified by a systematic search in PubMed, MEDLINE, Embase and Scopus, from database inception to 1 March 2021. Studies were included only if all the following conditions were met: DOR was defined using serum AMH concentration or AFC; miscarriage rate was reported separately for different groups of women categorized according to the AMH and/or AFC level; authors reported either the rate of intrauterine pregnancy loss before 22 weeks of gestation or enough data were available to calculate it. OUTCOMES From a total of 347 publications initially identified, 16 studies were included. Pooled results from 13 retrospective studies focusing on ART pregnancies showed a significantly higher rate of miscarriage in women with a low AMH, as compared to women with a medium or high serum AMH concentration (12 042 women, random effects model, odds ratio (OR) 1.35; 95% CI, 1.10–1.66; P = 0.004; I2=50%). The only prospective study on ART pregnancies failed to show any association (61 women, risk ratio (RR) 2.95; 95% CI, 0.66–3.18; P = 0.16). Data from two prospective studies, which included naturally conceived pregnancies, showed a significantly increased miscarriage risk for women with low serum AMH. However, these data could not undergo meta-analysis owing to differing study designs. Using three retrospective studies, we observed an association between low AFC and miscarriage incidence (three retrospective studies on ART pregnancies, random effects model, OR 1.81; 95% CI, 1.02–3.21; P = 0.04; I2=64%). WIDER IMPLICATIONS Our meta-analysis findings suggest that within the DOR patient subgroup, serum AMH and AFC biomarker levels may correlate with both the quantitative and qualitative aspects of ovarian reserve. However, owing to study limitations, the aetiology of this effect remains unclear and we are unable to define a causal relationship between DOR and increased miscarriage or to provide clinical recommendations based on this information. However, if confirmed by future well-designed studies, these findings would be profoundly informative for guiding women in family planning decisions.

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.

To test the hypothesis claiming an association between Human Papilloma Virus (HPV) sperm infection and idiopathic recurrent pregnancy loss (RPL).Multicenter retrospective case-control study.Three Italian University Hospitals.Cases included men belonging to couples affected by first trimester idiopathic RPL. Controls included men belonging to couples with proven fertility and no history of pregnancy loss. RPL was defined as the previous loss of two or more pregnancies. Couples were defined as 'fertile' if they achieved a full-term pregnancy within the year prior to enrollment in the study. All participants conceived without assistance.The association between HPV DNA sperm infection, as identified using polymerase chain reaction (PCR), and RPL.HPV DNA sperm infection was detected in 23 of 117 cases (20%; 95% confidence interval (CI) [13, 28%]) and in 3 of 84 controls (4%; 95% CI [1, 10%]) (p0.001). A comparison across baseline characteristics and multiple regression analysis did not identify any potentially confounding factors. Multivariate regression models showed a significant association between HPV DNA sperm infection and RPL (adjusted odds ratio (aOR) 7.44, 95% CI [2.08, 26.58], p=0.002 (Model 1); aOR 8.96, 95% CI [2.41, 33.44], p=0.001 (Model 2)).The prevalence of HPV sperm infection was significantly higher in couples affected by RPL than in their fertile counterparts. Notably, the semen sample was infected by HPV in approximately 1 out of 5 case-patients.

In vitro fertilization (IVF) is associated with a higher incidence of monozygotic twin pregnancies, which are known to be burdened by a higher risk of main obstetric complications. The reasons behind this association are still unclear. In the present study, we therefore investigate the risk factors for monozygotic twinning in pregnancies achieved by IVF. We conducted a multicenter retrospective case-control study. All IVF cycles performed between 2014 and 2019 at the infertility units of two Italian academic institutes were retrospectively reviewed. Only pregnancies obtained with single embryo transfer were eligible. A total of 50 monozygotic twin pregnancies (cases) were identified and matched in a 1:5 ratio to 250 singleton pregnancies (controls) by study center and study period. Monozygotic twin pregnancies were diagnosed by ultrasound. Women experiencing miscarriage could be included provided that the pregnancy loss occurred after a definitive diagnosis of monozygotic twin pregnancy. Demographic, clinical, and embryological characteristics were retrieved from patients' charts. Overall, the incidence of monozygotic twin pregnancies was 1.2% (50 out of 4016 single embryo transfers). At univariate analyses, statistically significant differences emerged for BMI, peripheral levels of estradiol and progesterone at the time of hCG administration, total number of retrieved suitable oocytes, freezing-thawing cycles, and assisted hatching. After performing a multivariate logistic analysis, only assisted hatching remained significantly associated with monozygotic twinning (adjusted odds ratio 2.32, 95%CI 1.03-5.25). Blastomere separation during the passage through this artificial hole or interference with the signaling pathway within the embryo could be the mechanisms involved.

Objective To investigate the impact of serum vitamin D level on in vitro fertilization (IVF) outcomes. Design Systematic review and meta-analysis. Setting Not applicable. Patients Infertile women undergoing conventional IVF or intracytoplasmic sperm injection (ICSI). Interventions Systematic search of PubMed, MEDLINE, EMBASE, Global Health, The Cochrane Library, Health Technology Assessment Database, and Web of Science from inception until July 2019 with cross-checking of references from relevant articles in English. Vitamin D levels were categorized into three groups: deficient ( 30 ng/mL). Before starting the data extraction, we registered the review protocol in PROSPERO (CRD42019134258). Main Outcome Measures We consider clinical pregnancy rate (CPR), live birth rate (LBR), and/or ongoing pregnancy rate (OPR) as primary outcomes. Likewise, the miscarriage rate was considered as a secondary outcome. Results Primary analysis showed that women with a replete level of vitamin D had higher CPR and LBR/OPR compared to those with a deficient of insufficient level of vitamin D. However, sensitivity analysis led to non-significant differences between the comparators for CPR (odds ratio 0.71, 95% confidence interval 0.47−1.08, I2 = 61%) and OPR/LBR (odds ratio 0.78, 95% confidence interval 0.56−1.08], I2 = 61%). Also, for miscarriage a statistically different rate was not reached. Conclusion Serum vitamin D levels do not influence IVF outcomes in terms of CPR, LBR/OPR, and miscarriage rate. Future large cohort studies are warranted to determine whether the threshold of vitamin D affects reproductive outcomes. Currently, there is a lack of consensus between the appropriate vitamin D threshold to predict reproductive outcomes compared to the one established for bone health. PROSPERO Number CRD42019134258.

Data on the effects of cancer treatments on fertility are conflicting. The aim of the present systematic review and meta-analysis was to determine the chances of childbirth in women survivors of different types of cancer. PubMed, MEDLINE, Embase and Scopus were searched from database inception to 17 July 2019 for published cohort, case-control and cross-sectional studies that investigated the reproductive chances in women survivors of different cancer types. Random-effects models were used to pool childbirth hazard ratios, relative risks, rate ratios and odds ratios, and 95% confidence intervals were estimated; 18 eligible studies were identified. Childbirth chances were significantly reduced in women with a history of bone cancer (HR 0.86, 95% CI 0.77 to 0.97; I2 = 0%; P = 0.02 (two studies); RaR 0.76, 95% CI 0.61 to 0.95; I2 = 69%; P = 0.01 (two studies); breast cancer (HR 0.74, 95% CI 0.61 to 0.90 (one study); RaR 0.51, 95% CI 0.47 to 0.57; I2 = 0%; P

The advent of random start protocols to shorten the time needed to store oocytes in women with malignancies has represented an important improvement in the field of fertility preservation. However, Randomized Controlled Trials are difficult to implement in this area and available evidence that supports this approach remains modest. To shed more light on this issue, we compared the follicular development between the ovary carrying the dominant follicle or the corpus luteum and the contralateral resting ovary in 90 women who underwent random start controlled ovarian stimulation (COS). In fact, ovarian response did not differ between the two ovaries. Subgroup analyses according to the phase of the cycle at the initiation of COS, the type of malignancy, the use of letrozole and the magnitude of the ovarian response did not allow to identify any condition showing a difference in the follicular response between the active and the resting ovaries. In conclusion, follicular growth does not seem to be perturbed by the presence of a dominant follicle or a corpus luteum.

Controlled ovarian hyperstimulation (COH) determines an anticipation of endometrial maturation and a premature occurrence of the implantation window, as shown by histological, histochemical, and molecular studies and indirectly by clinical trials. There is growing agreement that in patients hyper-responding to COH and in those undergoing transfer at the blastocyst stage, deferring the transfer in a subsequent frozen cycle could increase pregnancy outcomes. For blastocysts, implantation after a fresh transfer may be limited as the implantation window is already closed while, in hyper-responders to COH, the anticipation magnitude could be more marked thus hampering implantation also for cleavage-stage embryos. Research should focus in depth on pregnancy outcomes and on the most suitable modality to prepare the endometrium for frozen transfers.

Despite the fact that knowledge on obstetrical management of Inflammatory Bowel Diseases (IBDs) has greatly improved over the years, many patients still actively avoid pregnancy for fear of adverse maternal or neonatal outcomes, of adverse effects of pregnancy on the disease activity, of eventual IBD inheritance, or of an increased risk of congenital malformations. Indeed, though data prove that fertility is hardly affected by the disease, a reduced birth rate is nevertheless observed in patients with IBD. Misconceptions on the safety of drugs during gestation and breastfeeding may influence patient choice and negatively affect their serenity during pregnancy or lactation. Moreover, physicians often showed concerns about starting IBD medications before and during pregnancy and did not feel adequately trained on the safety of IBD therapies. IBD-expert gastroenterologists and gynecologists should discuss pregnancy and breastfeeding issues with patients when starting or changing medications in order to provide appropriate information; therefore, pre-conception counselling on an individualized basis should be mandatory for all patients of reproductive age to reassure them that maintaining disease remission and balancing the eventual obstetrical risks is possible.

Does the embryologist performing the embryo transfer impact the cycle outcome, in terms of ongoing pregnancy rate (OPR)?This single-centre retrospective study analysed the results, corrected for main confounders, from 28 embryologists and 32 physicians who performed respectively 24,992 and 24,669 fresh embryo transfers (either at cleavage or blastocyst stage) during a 20-year period from January 2000 to December 2019, in a university-affiliated tertiary care assisted reproductive technology (ART) centre. Primary outcome was OPR, defined as the number of viable pregnancies that had completed at least 12 weeks of gestation on the total number of embryo transfers performed. The study also assessed whether the embryologist's experience, measured in terms of number of embryo transfers performed prior to the day of the procedure, had an impact on their performance. The secondary aim was to assess which variable, between the embryologist and physician, more significantly impacted OPR.The overall unadjusted OPR was 22.54%. The embryologist performing the embryo transfer was found to significantly affect the OPR (P0.0001), corrected for potential confounders. However, the physician factor made a slightly greater contribution to the model (likelihood ratio 21.86, P0.001 versus likelihood ratio 17.20, P0.0001). No significant association was found between the experience of the embryologist and OPR (P = 0.067).These results show how the 'human factor' influences the chances of a positive outcome in the final step of a high-tech procedure and underline the importance of implementing an operator quality performance programme (both for physicians and embryologists) to ensure the maintenance of benchmark results and eventually retrain underperforming operators.

Most proposed definitions of recurrent implantation failure (RIF) are based on clinical judgement, probably affected by patients' demands. They are not based on robust statistical considerations. As a result, a diagnosis of RIF is commonly made too early, exposing couples to the risk of overdiagnosis and overtreatment. However, the situation is changing, and three statistical approaches have recently been proposed. The first is a probability model based on the chances of success per cycle and suggests for the definition three failed oocyte retrieval cycles with all embryos being transferred in women younger than 40 years of age. The second approach suggests an individualized diagnosis that takes into consideration multiple factors, while the third is also based on individualization but mainly relies on anticipated euploidy rates across the female age range. All these approaches have their pros and cons. Regardless of the specific peculiarities, they represent steps in the right direction, with the intent of providing a statistically sound definition. However, these attempts will not be useful unless endorsed by the scientific community in general. There is a pressing need for a rigorous and shared definition of RIF that will be widely accepted by researchers, scientific societies and other stakeholders, including patients.

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.

A consistent body of in vitro evidence supports a detrimental effect of endometriosis on ovarian steroidogenesis, in particular the synthesis of estrogens. However, clinical evidence is scanty and methodologically weak. This study aimed at clarifying whether peripheral 17-β-estradiol during IVF are influenced by the presence of endometriosis. Women undergoing IVF were retrospectively reviewed. Cases were women with a diagnosis of endometriosis. Controls were matched to cases in a 1:1 ratio by study period, age, total number of developed follicles on the day of hCG administration, protocol of hyperstimulation, gonadotropin used, and starting dose. The primary outcome was the ratio between serum levels of 17-β-estradiol and the total number of developed follicles. Fifty-three women with endometriosis and 53 controls were selected. The median ratio (interquartile range) between serum 17-β-estradiol and the total number of developed follicles in the two groups was 207 (164–282) and 201 (144–268) pg/ml, respectively (p = 0.46). Sensitivity analyses on the magnitude of the follicular response, the history of surgery for endometriomas, and the presence of endometriomas did not show any subgroup at increased risk of peripheral estrogens impairment. Endometriosis does not influence peripheral levels of 17-β-estradiol during IVF. Our findings argue against a biologically relevant effect of the disease on ovarian estrogen-synthesis.

Objective To evaluate whether telomere length (TL), mitochondrial-DNA (mt-DNA) or epigenetic age estimators based on DNA methylation (DNAm) pattern could be considered reliable predictors of in-vitro-fertilization (IVF) success in terms of live birth rate. Design Prospective cohort study Setting Infertility Unit of the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Patients 181 women aged 37–39 years who underwent IVF at a single-centre between January 2017 and December 2018. Interventions On the day of recruitment, blood samples were collected, and genomic DNA was isolated from white blood cells. TL, mt-DNA and DNAm assessment was performed using quantitative real-time polymerase chain reaction (qPCR). Biological age (DNAm age) was computed as the algorithm based on methylation pattern of five genes. Epigenetic age acceleration was estimated from the residuals of the linear model of epigenetic age regressed on chronological age. Long Interspersed Nuclear Elements (LINE)-1 methylation pattern was used as a surrogate for global DNA methylation. Main outcome measures This study investigated whether peripheral TL, mt-DNA and DNAm could predict live birth in IVF cycles. Results TL, mt-DNA and LINE-1 methylation were not associated with IVF success. Conversely, DNAm age resulted significantly lower in women who had a live birth compared to women who did not (36.1 ± 4.2 and 37.3 ± 3.3 years, respectively, p = 0.04). For DNAm age, odds ratio (OR) for live birth per year of age was 0.90 (95%CI: 0.82–0.99, p = 0.036) after adjusting for FSH and antral follicle count (AFC) and 0.90 (95%CI: 0.82–0.99, p = 0.028) after adjusting also for number of oocytes retrieved. A significant association also emerged for epigenetic age acceleration after adjustments (OR = 0.91, 95%CI: 0.83–1.00, p = 0.048). Conclusion DNAm age is associated with IVF success but the magnitude of this association is insufficient to claim a clinical use. However, our findings are promising and warrant further investigation. Assessment of biological age using different epigenetic clocks or focusing on different tissues may reveal new predictors of IVF success.

STUDY QUESTION Is there an association between the different endometrial preparation protocols for frozen embryo transfer (FET) and obstetric and perinatal outcomes? SUMMARY ANSWER Programmed FET protocols were associated with a significantly higher risk of hypertensive disorders of pregnancy (HDP), pre-eclampsia (PE), post-partum hemorrhage (PPH) and cesarean section (CS) when compared with natural FET protocols. WHAT IS KNOWN ALREADY An important and growing source of concern regarding the use of FET on a wide spectrum of women, is represented by its association with obstetric and perinatal complications. However, reasons behind these increased risks are still unknown and understudied. STUDY DESIGN, SIZE, DURATION Systematic review with meta-analysis. We systematically searched PubMed, MEDLINE, Embase and Scopus, from database inception to 1 November 2021. Published randomized controlled trials, cohort and case control studies were all eligible for inclusion. The risk of bias was assessed using the Newcastle–Ottawa Quality Assessment Scale. The quality of evidence was also evaluated using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. PARTICIPANTS/MATERIALS, SETTING, METHODS Studies were included only if investigators reported obstetric and/or perinatal outcomes for at least two of the following endometrial preparation protocols: programmed FET cycle (PC-FET) (i.e. treatment with hormone replacement therapy (HRT)); total natural FET cycle (tNC-FET); modified natural FET cycle (mNC-FET); stimulated FET cycle (SC-FET). MAIN RESULTS AND THE ROLE OF CHANCE Pooled results showed a higher risk of HDP (12 studies, odds ratio (OR) 1.90; 95% CI 1.64–2.20; P LIMITATIONS, REASONS FOR CAUTION The principal limitation concerns the heterogeneity across studies in: (i) timing and dosage of HRT; (ii) embryo stage at transfer; and (iii) inclusion of preimplantation genetic testing cycles. To address it, we undertook subgroup analyses by pooling only ORs adjusted for a specific possible confounding factor. WIDER IMPLICATIONS OF THE FINDINGS Endometrial preparation protocols with HRT were associated with worse obstetric and perinatal outcomes. However, because of the methodological weaknesses, recommendations for clinical practice cannot be made. Well conducted prospective studies are thus warranted to establish a safe endometrial preparation strategy for FET cycles aimed at limiting superimposed risks in women with an ‘a priori’ high-risk profile for obstetric and perinatal complications. STUDY FUNDING/COMPETING INTEREST(S) None. REGISTRATION NUMBER CRD42021249927.

Objective To investigate the impact of controlled ovarian hyperstimulation (COH) for in vitro fertilization (IVF) on thyroid function. Design Systematic review and meta-analysis. Setting Not applicable. Patient(s) Infertile women undergoing conventional IVF or intracytoplasmic sperm injection. Intervention(s) Systematic search of PubMed, MEDLINE, Embase, Scopus, and Web of Science from inception until November 2020. Studies could be included only if they met the following criteria: subjects were classified as euthyroid or hypothyroid; serum thyroid-stimulating hormone (TSH) and/or free thyroxine (FT4) levels were evaluated before COH; and the same thyroid function test was reassessed after COH (i.e., at the time of trigger for final follicle maturation and/or at pregnancy test). Main Outcome Measure(s) Mean difference (MD) between the serum TSH or FT4 levels assessed after COH and before COH. Result(s) In euthyroid women, the serum TSH levels assessed at the time of trigger and at the time of pregnancy test were significantly higher than those at baseline (MD: 0.69 mIU/L, 95% confidence interval [CI]: 0.30–1.08, I2 = 93% and MD: 0.67, 95% CI: 0.49–0.85, I2 = 72%, respectively). The serum FT4 levels did not undergo significant changes. Subanalysis confirmed an increase in the TSH level after restricting the analysis to women treated with gonadotropin-releasing hormone agonist protocols and to those who achieved pregnancy. A pronounced increase in the TSH level was observed in women treated for hypothyroidism (MD: 1.50 mIU/L, 95% CI: 1.10–1.89, I2 = 0%). Conclusion(s) Pooling of the results showed a significant increase in serum TSH level in women undergoing COH for IVF. This change was particularly pronounced in women treated for hypothyroidism. New thyroid function screening strategies for women undergoing COH are warranted.

The aim of the present systematic review and meta-analysis was to assess the effect of the different therapeutic options for repeated embryo implantation failure (RIF) on a subsequent IVF cycle outcome. Twenty-two RCTs and nineteen observational studies were included. Pooling of results showed a beneficial effect of intrauterine PBMC infusion on both CPR (RR 2.18; 95% CI 1.58–3.00; p

In vitro fertilization (IVF) is associated with a higher incidence of monozygotic twin pregnancies, which are known to be burdened by a higher risk of main obstetric complications. The reasons behind this association are still unclear. In the present study, we therefore investigate the risk factors for monozygotic twinning in pregnancies achieved by IVF. We conducted a multicenter retrospective case-control study. All IVF cycles performed between 2014 and 2019 at the infertility units of two Italian academic institutes were retrospectively reviewed. Only pregnancies obtained with single embryo transfer were eligible. A total of 50 monozygotic twin pregnancies (cases) were identified and matched in a 1:5 ratio to 250 singleton pregnancies (controls) by study center and study period. Monozygotic twin pregnancies were diagnosed by ultrasound. Women experiencing miscarriage could be included provided that the pregnancy loss occurred after a definitive diagnosis of monozygotic twin pregnancy. Demographic, clinical, and embryological characteristics were retrieved from patients' charts. Overall, the incidence of monozygotic twin pregnancies was 1.2% (50 out of 4016 single embryo transfers). At univariate analyses, statistically significant differences emerged for BMI, peripheral levels of estradiol and progesterone at the time of hCG administration, total number of retrieved suitable oocytes, freezing-thawing cycles, and assisted hatching. After performing a multivariate logistic analysis, only assisted hatching remained significantly associated with monozygotic twinning (adjusted odds ratio 2.32, 95%CI 1.03-5.25). Blastomere separation during the passage through this artificial hole or interference with the signaling pathway within the embryo could be the mechanisms involved.

Several pathogenetic mechanisms have been postulated to explain the association between endometriosis and subfertility. However, to date, definitive conclusions cannot be drawn. In this study, we hypothesized that the reduced exploitation of the natural chances of conception could be an additional detrimental factor. Due to dyspareunia or the need for hormonal treatment to temper pelvic pain, one may expect affected women to exploit less the chances of natural pregnancy. In this cross-sectional study of 292 women undergoing IVF, we investigated the severity of pelvic pain symptoms, the sexual function (using the Female Sexual Function Index [FSFI]), and the reproductive strategies of women with (n = 62) and without (n = 230) endometriosis. Basal clinical and demographic characteristics did not differ between the two groups. Conversely, all pelvic pain symptom scores were increased in women with endometriosis. Endometriosis patients also showed greater pain at the FSFI, but no other significant differences were detected as regards sexual function. The use of hormonal contraceptive agents did not differ between the study groups. Moreover, questions aimed at disentangle whether affected women exploited less the natural chances of pregnancy did not reveal any significant difference. In conclusion, this study does not support the hypothesis that women with endometriosis exploit less the chances of natural pregnancy, despite the presence of pelvic pain.

A consistent body of in vitro evidence supports a detrimental effect of endometriosis on ovarian steroidogenesis, in particular the synthesis of estrogens. However, clinical evidence is scanty and methodologically weak. This study aimed at clarifying whether peripheral 17-β-estradiol during IVF are influenced by the presence of endometriosis. Women undergoing IVF were retrospectively reviewed. Cases were women with a diagnosis of endometriosis. Controls were matched to cases in a 1:1 ratio by study period, age, total number of developed follicles on the day of hCG administration, protocol of hyperstimulation, gonadotropin used, and starting dose. The primary outcome was the ratio between serum levels of 17-β-estradiol and the total number of developed follicles. Fifty-three women with endometriosis and 53 controls were selected. The median ratio (interquartile range) between serum 17-β-estradiol and the total number of developed follicles in the two groups was 207 (164-282) and 201 (144-268) pg/ml, respectively (p = 0.46). Sensitivity analyses on the magnitude of the follicular response, the history of surgery for endometriomas, and the presence of endometriomas did not show any subgroup at increased risk of peripheral estrogens impairment. Endometriosis does not influence peripheral levels of 17-β-estradiol during IVF. Our findings argue against a biologically relevant effect of the disease on ovarian estrogen-synthesis.

In the last decade, testosterone replacement therapy (TRT) has been increasingly prescribed to treat a controversial condition known as 'late-onset hypogonadism (LOH)'. This syndrome is diagnosed in men who, for no discernible reason other than older age, obesity or ill health have serum testosterone concentrations below the normal range for healthy young men and report one or more of the following symptoms: muscle weakness or wasting, mood, behaviour and cognition-related symptoms and sexual function or libido impairment. However, recent evidence has demonstrated that testosterone drugs do not substantially ameliorate these symptoms and, more worryingly, that their long-term use may be associated with severe adverse effects (i.e. increased risk of prostate cancer, stroke and myocardial infarction, worsening of benign prostatic hyperplasia symptoms and testicular atrophy). Nonetheless, testosterone drugs have exhibited extraordinary commercial success and their pharmaceutical sales are steadily rising. Behind this apparently unjustifiable trend there are deliberate, well designed direct and indirect pharmaceutical marketing initiatives that exploit the conviction rooted in contemporary society that testosterone can reverse the effects of ageing and ensure social accomplishment. Commercial mechanisms have laid the foundation for disease mongering of LOH and also have resulted a considerable expansion of the indications for treatment. This promotion model deserves particular attention since it is applicable to any drug with a purportedly favourable risk-benefit ratio not supported by evidence.

The clinical management of women with deep peritoneal endometriosis remains controversial. The debate focuses mainly on the precise role of hormonal medical treatment and surgery and on the most suitable surgical technique to be used. In particular, considering the risks of second-line surgery, prevention of recurrences after first-line surgery is a priority in this context. Post-surgical medical therapy has been advocated to improve the effectiveness of surgery and prevent recurrences. However, adjuvant therapy, i.e. a short course of 3-6 months of hormonal therapy after surgery, has been proven to be of limited or no benefit for endometriosis in general and for deep peritoneal endometriosis in particular. On the other hand, two cohort studies suggest a beneficial effect of prolonged hormonal therapy after surgery for deep endometriosis. Even if this evidence is too weak to confidently advocate systematic administration of prolonged medical therapy after surgery, we argue in favour of this approach because of the strong association of deep endometriosis with other disease forms. In fact, women operated on for deep endometriosis may also face recurrences of endometriomas, superficial peritoneal lesions and pelvic pain in general. The demonstrated high effectiveness of prolonged postoperative therapy for the prevention of endometriomas' formation and dysmenorrhea recurrence should thus receive utmost consideration in the decision-making process.