Several studies have demonstrated that the soil of public parks presents an important source of infection which has a significant impact on public health. Children are the main group affected by accidentally ingestion of contaminated soil. This study was performed in order to identify the presence of zoonotic parasites in dog and cat faecal and soil samples from public parks of Madrid, Spain. Six hundred twenty-five and seventy-nine soil and faecal samples (presumably from dogs and cats) respectively were collected from 67 parks. Intestinal parasites were identified in 27 parks (40.3%), which were contamined with Giardia sp. (19.4%), microsporidia (19.4%), Toxocara spp. (16.4%), Cryptosporidium sp. (6%), Entamoeba histolytica (3%) and Ancylostomidae (3%). Combinations of two or more intestinal parasites were found in 11 parks, and it was common to find Giardia and microsporidia together in samples. Intestinal parasites were detected in 18% (112/625) of soil samples. The most frequent parasite species found in the examined soil samples were Toxocara spp. (16.4%), followed by Giardia sp. (4.5%) and Strongyloides sp. larvae (3%). The zoonotic parasites found in the 79 faecal samples were Giardia sp. (17.7%), Cryptosporidium sp. (9%), E. histolytica (2.5%), Trichuris vulpis (1.3%), Toxascaris leonina (1.3%) and microsporidia spores (28%). Microsporidia characterization by amplification of DNA confirmed 10 samples as positive, eight for E. bieneusi and two for E. hellem by PCR. The role of those parasites in the environment are discussed.

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STUDY QUESTION Does an individualised luteal phase support (iLPS), according to serum progesterone (P4) level the day prior to euploid frozen embryo transfer (FET), improve pregnancy outcomes when started on the day previous to embryo transfer? SUMMARY ANSWER Patients with low serum P4 the day prior to euploid FET can benefit from the addition of daily subcutaneous P4 injections (Psc), when started the day prior to FET, and achieve similar reproductive outcomes compared to those with initial adequate P4 levels. WHAT IS KNOWN ALREADY The ratio between FET/IVF has spectacularly increased in the last years mainly thanks to the pursuit of an ovarian hyperstimulation syndrome free clinic and the development of preimplantation genetic testing (PGT). There is currently a big concern regarding the endometrial preparation for FET, especially in relation to serum P4 levels around the time of embryo transfer. Several studies have described impaired pregnancy outcomes in those patients with low P4 levels around the time of FET, considering 10 ng/ml as one of the most accepted reference values. To date, no prospective study has been designed to compare the reproductive outcomes between patients with adequate P4 the day previous to euploid FET and those with low, but restored P4 levels on the transfer day after iLPS through daily Psc started on the day previous to FET. STUDY DESIGN, SIZE, DURATION A prospective observational study was conducted at a university-affiliated fertility centre between November 2018 and January 2020 in patients undergoing PGT for aneuploidies (PGT-A) IVF cycles and a subsequent FET under hormone replacement treatment (HRT). A total of 574 cycles (453 patients) were analysed: 348 cycles (leading to 342 euploid FET) with adequate P4 on the day previous to FET, and 226 cycles (leading to 220 euploid FET) under iLPS after low P4 on the previous day to FET, but restored P4 levels on the transfer day. PARTICIPANTS/MATERIALS, SETTING, METHODS Overall we included 574 HRT FET cycles (453 patients). Standard HRT was used for endometrial preparation. P4 levels were measured the day previous to euploid FET. P4 > 10.6 ng/ml was considered as adequate and euploid FET was performed on the following day (FET Group 1). P4 < 10.6 ng/ml was considered as low, iLPS was added in the form of daily Psc injections, and a new P4 analysis was performed on the following day. FET was only performed on the same day when a restored P4 > 10.6 ng/ml was achieved (98.2% of cases) (FET Group 2). MAIN RESULTS AND THE ROLE OF CHANCE Patient’s demographics and cycle parameters were comparable between both euploid FET groups (FET Group 1 and FET Group 2) in terms of age, weight, oestradiol and P4 levels and number of embryos transferred. No statistically significant differences were found in terms of clinical pregnancy rate (56.4% vs 59.1%: rate difference (RD) −2.7%, 95% CI [−11.4; 6.0]), ongoing pregnancy rate (49.4% vs 53.6%: RD −4.2%, 95% CI [−13.1; 4.7]) or live birth rate (49.1% vs 52.3%: RD −3.2%, 95% CI [−12; 5.7]). No significant differences were also found according to miscarriage rate (12.4% vs 9.2%: RD 3.2%, 95% CI [−4.3; 10.7]). LIMITATIONS, REASONS FOR CAUTION Only iLPS through daily Psc was evaluated. The time for Psc injection was not stated and no serum P4 determinations were performed once the pregnancy was achieved. WIDER IMPLICATIONS OF THE FINDINGS Our study provides information regarding an ‘opportunity window’ for improved ongoing pregnancy rates and miscarriage rates through a daily Psc injection in cases of inadequate P4 levels the day previous to FET (P4 < 10.6 ng/ml) and restored values the day of FET (P4 > 10.6 ng/ml). Only euploid FET under HRT were considered, avoiding one of the main reasons of miscarriage and implantation failure and overcoming confounding factors such as female age, embryo quality or ovarian stimulation protocols. STUDY FUNDING/COMPETING INTEREST(S) No external funding was received. B.C. reports personal fees from MSD, Merck Serono, Ferring Pharmaceuticals, IBSA and Gedeon Richter outside the submitted work. N.P. reports grants and personal fees from MSD, Merck Serono, Ferring Pharmaceuticals, Theramex and Besins International and personal fees from IBSA and Gedeon Richter outside the submitted work. The remaining authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER NCT03740568.

STUDY QUESTION Are progesterone (P) levels on the day before natural cycle frozen embryo transfer (NC-FET) associated with live birth rate (LBR)? SUMMARY ANSWER Regular ovulatory women undergoing NC-FET with serum P levels <10 ng/ml on the day before blastocyst transfer have a significantly lower LBR than those with serum P levels >10 ng/ml. WHAT IS KNOWN ALREADY The importance of serum P levels around the time of embryo transfer in patients undergoing FET under artificial endometrial preparation has been well established. However, no study has analyzed the importance of serum P levels in patients undergoing FET under a true natural endometrial preparation cycle. STUDY DESIGN, SIZE, DURATION This was a retrospective cohort study including 294 frozen blastocyst transfers under natural cycle endometrial preparation at a university-affiliated fertility centre between January 2016 and January 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS All patients had regular menstrual cycles and underwent NC-FET with their own oocytes. Only patients who had undergone serum P measurement between 8 am and 11 am on the day before FET were included. Patients did not receive any external medication for endometrial preparation or luteal phase support. Patients were divided into two groups according to serum P levels below or above 10 ng/ml on the day before FET. Univariate analysis was carried out to describe and compare the cycle characteristics with reproductive outcomes. To evaluate the effect of P, a multivariable logistic model was fitted for each outcome after adjusting for confounding variables. MAIN RESULTS AND THE ROLE OF CHANCE Mean serum P levels on the day before FET were significantly higher in patients who had a live birth compared to those who did not (14.5 ± 7.0 vs 12.0 ± 6.6 ng/ml, 95% CI [0.83; 4.12]). The overall clinical pregnancy rate (CPR) and LBR were 42.9% and 35.4%, respectively. Patients in the higher P group (>10 ng/ml) had a higher LBR (41.1% vs 25.7%: risk difference (RD) 15.4%, 95% CI [5; 26]) and CPR (48.6% vs 33.0%: RD 15.6%, 95% CI [4; 27]). Patients with higher serum P levels on the day before FET (63% of patients) had an improved LBR (odds ratio: 1.05; 95% CI [1.02; 1.09]). Women with serum P levels <10 ng/ml on the day before FET (37% of patients) had significantly higher weights (62.5 ± 9.9 vs 58.1 ± 7.1 kg, 95% CI [1.92; 6.90]) and BMI (22.9 ± 3.6 vs 21.6 ± 2.7 kg/m2, 95% CI [0.42; 2.25]) compared to patients with P levels >10 ng/ml. LIMITATIONS, REASONS FOR CAUTION The main limitation of our study is its retrospective design. Other potential limitations are the detection of LH surge through urine testing and the inclusion of patients who did and did not undergo preimplantation genetic testing for aneuploidies. The protocol used in our institution for monitoring NC-FET does not look for the onset of progesterone secretion by the corpus luteum, and a slow luteinisation process or delay of corpus luteum function cannot be ruled out. WIDER IMPLICATIONS OF THE FINDINGS We provide evidence that a minimum serum P threshold (P >10 ng/ml) might be required for improved reproductive outcomes in NC-FET. This result suggests that there are different mechanisms by which P is produced and/or distributed by each patient. This study also provides an excellent model to evaluate the impact of luteal phase defect through NC-FET. A prospective evaluation to assess whether P supplementation should be individualised according to patient’s needs is necessary to support our findings. STUDY FUNDING/COMPETING INTEREST(S) No external funding was used, and there are no competing interests.

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