Objective: An increased risk of preeclampsia has been described when performing donor intrauterine insemination. One of the possible causes is immunological tolerance to semen. Both lesbian and single women, who have theoretically lower exposure to seminal fluid than heterosexual women requesting artificial insemination with donor, sperm should have a higher incidence of preeclampsia, although this fact has not been analyzed previously. Methods: The population under study consisted of 439 gestations <24 weeks, achieved with artificial insemination with donor sperm, performed in two different populations: heterosexual couples and SLTG (single women, lesbian women, women with transgender partner). Preeclampsia rates and other perinatal outcomes were compared. Results: No significant differences were found in the development of preeclampsia in the SLTG group 6.03% (12/199) vs. heterosexual patients 5.83% (14/240), p=0.93. In the SLTG group, compared with heterosexual group, age (36 vs. 35, p=0.002) and BMI (24.8 vs. 23.6, p=0.002) were somewhat higher. After adjusting for BMI, age and multiplicity of gestation, there were no significant differences in the risk of preeclampsia between SLTG group and heterosexual women. As expected, in women with preeclampsia, gestational ages and newborn weights (quantitative markers of preeclampsia severity) were significantly lower than in women without preeclampsia. Conclusions: Our study suggests that neither sexual orientation nor previous sperm exposure carries a higher risk of preeclampsia, especially after correction for age and weight. Artificial insemination with donor sperm performed on single women, lesbians or women with transgender partners is a safe, simple and efficient technique, just as in heterosexual couples.

Purpose:To ascertain whether ovarian hyperresponse and oocyte vitrification (OV) are associated with an increase in embryo aneuploidy. Methods:Retrospective study involving 197 629 retrieved oocytes. We studied the euploidy rate (ER), the ER according to the number of retrieved oocytes (ERaRO) and according to the number of metaphase II oocytes (MIIO) (ERaMII), and the quotient "MIIO/retrieved oocytes" or mature oocyte rate (MOR). Results:The maximal ERaRO corresponded when only one oocyte was recovered (27% ± 44%), and was somewhat lower when two oocytes were retrieved (14% ± 31%). From the 3rd oocyte until the 36th oocyte, the ERaRO remained constant at around 10%. Regarding MIIO, a similar pattern was observed. For a given number of oocytes, usually the ER was higher if MOR was 100%. Moreover, even when considering only cases with a 100% MOR, the ER was significantly higher in cases where there were 1-2 MIIO. Concerning OV, the regression analysis after adjustment showed that vitrified oocytes had a similar rate of aneuploidy to fresh oocytes. Conclusions:Both ovarian hyperresponse and OV are safe procedures concerning embryo euploidy. The better ER obtained, especially in cases with 1-2 oocytes, opens a new line of research concerning the superior oocyte quality from the point of view of the euploidy of cycles with 1-2 oocytes.

Abstract Background Delineating the cellular origins of extracellular vesicles (EVs) enables the detection of clinically relevant changes in dynamic and complex tissues, such as the endometrium, which are not characterizable through single biomarker assays. Transcriptome deconvolution into cellular composition using deep learning methods provides a means to explore this complexity. However, such computational methods have not been previously applied to EV bulk transcriptomes, and their efficacy in profiling EV population changes and concordance to tissue throughout the menstrual cycle remains unknown. Methods This observational cross-sectional study utilized a deconvolutional generative deep learning algorithm, BulkTrajBlend, trained on a comprehensive human endometrial single-cell RNA sequencing (scRNA-seq) atlas. The model was applied to deconvolve paired bulk transcriptomes from endometrial tissue and uterine fluid EVs (UF-EVs) across the proliferative (P, n=4), early-secretory (ES, n=5), mid-secretory (MS, n=5), and late-secretory (LS, n=5) phases from healthy, fertile women. To validate generalizability, independent UF-EV datasets (ES, n=12; MS, n=12) obtained via different laboratory protocols were included. Deconvolved pseudo-single-cell (pSC) profiles from UF-EV data were subsequently integrated with Visium spatial transcriptomics slides of human endometrium (P, n=2; MS, n=4; ES, n=2). Results We developed a foundation model-based approach utilizing self-supervised learning to determine the cellular origin of EVs from their transcriptomic profiles. By mapping the generated pSC profiles to spatial transcriptomic data, we evaluated spatial origins of EVs. The statistical analysis demonstrated that UF-EV transcriptome deconvolution reflects the dynamic changes in the cellular composition of endometrial tissue across the menstrual cycle phases. The ability to distinguish accurately between proliferative and decidualizing menstrual cycle phases (ROC-AUC = 0.98) using cellular profile of deconvoluted UF-Evs transcriptome enables non-invasive profiling of endometrial tissue. Conclusions Our findings indicate the feasibility of determining endometrial tissue cellular composition using UF-EV transcriptomics. This methodology enables refined, non-invasive endometrial testing, avoiding invasive biopsy procedures. Based on deconvolution results, we are able to correlate UF-EV content to tissue, and distinguish between menstrual cycle phases. These results build toward a multifactorial screening method for abnormalities within the endometrium.

To study the influence of parental age on aneuploidy rates (AR) in PGT-A cycles and on the recurrence rate. A total of 16,029 PGT-A cycles were studied over a 9-year period. The median age was 40.0 [37.0; 41.0] in women and 40.0 [37.0; 43.0] in men. In 48.3

Objectives: To assess if the instillation of plasma rich growth factors (PRGFs) improves endometrial thickness (EMT) in frozen embryo cycles performed under hormone treatment where the endometrium was very thin (≤5 mm). Methods: First, a randomized controlled trial (RCT) was performed comparing women only receiving an increase in estrogen therapy (n = 9) and women receiving both the increase in estrogen therapy and three instillations of PRGF (n = 13). The second part of the study consisted of a prospective observational follow-up of the patients included in the RCT (for 1–3 months in the study group, and for 1–6 months in the control group). Results: In the RCT, there was an increase in EMT in both the PRGF and control groups. However, the increase was significantly higher in the PRGF group (1.30 ± 0.67 mm) compared to the control group (0.58 ± 0.51 mm). In the PRGF group, 23% achieved an EMT of 7 mm compared to 0% in the control group. There were 2 pregnancies in the PRGF group resulting from the 3 transfers performed in that group. No transfer was carried out in the control group. There was a significant increase in EMT 1–3 months after the PRGF cycle. The live birth rate per transfer was 20% in the cycles following the PRGF cycles, whereas it was 30% per starting woman. For women in the control group who later underwent PRGF, the LBR was 57.1%. Conclusions: PRGF instillation in cases of very thin endometrium increases EMT moderately. It is suggested that the beneficial effect of PRGF may persist for 1–3 cycles after instillation.

Klinefelter Syndrome (KS) is an aneuploid genetic condition in males characterized by at least one additional copy of the X chromosome. Due to fibrotic degeneration of the testis, these patients suffer infertility in the future. The pathogenic mechanism by which this occurs is still not well known. Here, we establish three Induced Pluripotent Stem cell (iPSC) lines derived from prepuberal and peripuberal KS patient's testicular somatic cells. These could be the first steps towards the creation of an in vitro platform to analyze the molecular mechanisms underlying pathophysiology of the testes in KS patients.

Introduction Scientific evidence has shown that regular physical activity (PA) is beneficial to health. However, no consensus has been reached on the association between PA and the success rates of assisted reproduction treatments. The purpose of the present study was to determine whether various levels of physical activity have an influence on ovarian response to controlled stimulation in 'in vitro fertilization' (IVF) or 'intracytoplasmic sperm injection' (ICSI) cycles, defined as number of retrieved and mature oocytes. Methods This prospective observational study included 617 infertile women undergoing IVF/ICSI cycles between January 2019 and October 2020. PA was assessed prior to the IVF cycle, using the International Physical Activity Questionnaire short form (IPAQ) and triaxial accelerometers. Patients were classified into three groups: low, moderate and high PA. Results Globally, the number of retrieved oocytes was similar in all three groups according to IPAQ (9.23 ± 7.72; 8.35 ± 5.57; 8.82 ± 6.38). Something similar happened with the number of mature oocytes (6.97 ± 5.99; 6.84 ± 4.85; 7.05 ± 5.61). PA did not influence the number of oocytes (retrieved or mature) in most clinical subgroups established (age, smoking, body mass index (BMI), anti-mullerian hormone (AMH)). However, in the subgroup 'endometriosis' the number of mature oocytes was significantly superior in high and moderate vs low PA (p=0.024). In the subgroup 'ovulatory disorders' there were also more mature oocytes in high and moderate vs low PA (p=0.038). When performing the analysis according to accelerometer there were globally no significant differences between PA groups, nor in most clinical subgroups considered. Only in women with normal BMI, high PA was significantly associated with a greater number of collected (p=0.005) and mature oocytes (p=0.004). Conclusion Globally, PA had no influence on ovarian response in IVF cycles, defined as number of retrieved and mature oocytes. However, in certain clinical subgroups (endometriosis, ovulatory disorders, normal-BMI) high PA was associated with a superior ovarian response.

The transcription factor GATA4 is found in Sertoli and Leydig cells, whereas SOX9 is exclusive to Sertoli cells, being both factors essential for the normal development of murine and human fetal testis. In turn, the steroidogenic acute regulatory protein (STAR) is specifically expressed in Leydig cells. Nevertheless, the function of STAR, GATA4 and SOX9 in peripubertal, adolescent and adult testes in Klinefelter syndrome and azoospermic patients remains poorly understood. To characterize the developmental expression of STAR, GATA4 and SOX9 in human testicular somatic cells, we performed immunofluorescence using fetal, peripubertal, adolescent and adult testes. Our findings demonstrate that STAR is absent in early fetal stages, but present in Leydig cells from 12 weeks of gestation, as well as in peripubertal, adolescent and adult Klinefelter patients, in the adult testis with idiopathic azoospermia and in men showing normal spermatogenesis. GATA4 was expressed in both Sertoli and Leydig cells during all the studied developmental stages and in peripubertal, adolescent and adult patients with and without spermatogenesis. SOX9 was mainly expressed in Sertoli cells in fetal, peripubertal, adolescent and adult Sertoli cell patients. In patients with Klinefelter syndrome as well as in men with or without spermatogenesis SOX9 was also found in Leydig cells. Our findings support the premise that STAR is a key steroidogenic protein for androgen development in the fetal testis, that GATA4 regulates Sertoli and Leydig cells during testis development and that SOX9 regulates the development of Sertoli cells and is present in the Leydig cells of patients with azoospermia.

Research questionWhat is the influence of biological, technical and clinical factors on PGT-A embryo outcomes and what is the recurrence pattern?DesignRetrospective study including 64,701 embryos undergoing PGT-A in the same laboratory between 2011 and 2019. Biopsies were performed at the day 3 embryo stage (48.3%) or blastocyst stage (51.7%). Advanced maternal age (AMA) was the main indication (65.6%).ResultsThe aneuploidy rate (AR) was 67.8%, higher in women >35 years than in ≤35 years (71.8% vs 47.4%), and higher in day 3 embryo vs blastocyst biopsies (77.50% vs 58.60%). The trisomy/ monosomy ratio was 1.0 on blastocysts vs 0.8 in day 3 embryos. Trisomy 21 was present in 4.9% of the embryos. In aneuploid embryos, the probability of having ≥1-involved chromosome followed a decreasing exponential pattern. The probability of an embryo being was constant around 30% (40% in blastocysts, 20% in day 3 embryos). The cumulative probability of having ≥1 euploid embryo after 10 biopsied embryos was 95% in blastocysts and 80% in day-3 embryos. AMA was associated with a much higher AR than all other indications, which among them had similar AR and chromosomal involvement.ConclusionsThere is a considerable decrease in AR from blastocysts to day 3 embryos, which is most notable for monosomies. While AMA shows an increase in AR and a specific chromosomal pattern of involvement, the remaining indications showed a similar AR and chromosomal pattern. Even after producing many consecutive aneuploid embryos, the possibility of obtaining a euploid embryo is not negligible.

RESEARCH QUESTION:What is the fecundity rate among fertile couples, and which factors influence it?DESIGN:Retrospective study of all puerperae attending Cruces University Hospital Human Reproduction Unit over 9 months. An anonymous questionnaire was circulated to all patients, and 2510 valid completed questionnaires were collected. The main inclusion criterion was natural conception resulting in delivery. Pregnancies resulting from ART and contraceptive method failure were excluded. Investigated parameters were time to pregnancy, age and smoking (in women and men), previous pregnancies and intercourse frequency. A mathematical formula was developed to predict the per-month fecundity rate (PMFR).RESULTS:The cumulative fecundity rate was 29.08%, 54.26%, 68.61%, 89.88%, 96.95% and 98.63% (at 1, 3, 6, 12, 24 and 36 months); between 12 and 36 months, the average PMFR ranged from 8.53-7.48%. Only 1.68% of pregnancies occurred between 24 and 36 months, and only 1.37% thereafter. The best fecundity markers were obtained in the group who had sexual intercourse seven to eight times a week. Women and men younger than 25 years had lower fecundity markers than those aged between 25 and 40 years.CONCLUSIONS:Fertile couples have a non-negligible per-month fecundity rate between 12 and 36 months, which should be considered when planning fertility studies. The lower fecundity rate observed in women and men aged younger than 25 years deserves more study. Coital frequencies of more than two or three times a week did not affect the fecundity rate and was better with frequencies of seven to eight times a week.

OBJECTIVE:The aim of our study was to assess if the addition of PRGF to healthy human sperm affects its motility and vitality.METHODS:This was a prospective study, with 44 sperm donors on whom sperm analysis was performed. Nine mL of blood was collected and PRGF was obtained using PRGF-Endoret® technology. The influence of different dilutions of PRGF (5%, 10%, 20%, 40%) applied to 15 sperm donors was compared, and sperm motility was assessed after 30 minutes. In the second part of the study, 29 sperm donors were studied to analyze the influence of 20% dilution of PRGF at 15, 30 and 45 minutes in fresh and thawed sperm samples. Motility was assessed after the addition of PRGF and after analysis each aliquot was frozen. After thawing, concentration and motility were assessed at the same time periods.RESULTS:There were no differences in sperm motility in fresh samples between dilutions of PRGF when assessed 30 minutes after administration, nor between them, nor when compared to the control group immediately prior to treatment. No trend was observed between motility and PRGF dilution in linear regression analysis. There were no significant differences in thawed samples.CONCLUSIONS:The administration of 20% PRGF dilution had no effect on sperm motility compared to samples without PRGF. In addition, there was no change in sperm vitality when comparing samples with and without PRGF. More studies focusing on subnormal sperm samples, analyzing different PRGF concentrations and increasing the number of study variables are needed.

OBJECTIVE:Endogenous opioid peptides were reported to be involved in the regulation of reproductive physiology and their precursors and receptors were described in many of the male and female reproductive tissues. Mu opioid receptor (MOR) was described in human endometrial cells and its expression and localization changed during the menstrual cycle. However, there is no data from the distribution of the other opioid receptors: Delta (DOR) and Kappa (KOR). The objective of the present work was to analyze the dynamics of expression and localization of DOR and KOR in human endometrium throughout the menstrual cycle.STUDY DESIGN:Human endometrial samples from different menstrual cycle phases were analyzed by immunohistochemistry.RESULTS:DOR and KOR were present in all samples analyzed and the protein expression and localization changed throughout the menstrual cycle. Both receptor expression increased during the late proliferative phase and decreased during the late secretory-one, especially in the luminal epithelium. DOR expression was generally higher than KOR expression in all cell compartments.CONCLUSIONS:The presence of DOR and KOR in human endometrium and their dynamic changes during the menstrual cycle join the results previously obtained in MOR suggesting a possible role of opioids in reproduction events related to the human endometrium.

ABSTRACT Research question What is the fecundity rate among fertile couples and which factors have an influence on it? Design Retrospective study by means of an anonymous questionnaire provided to all the puerperae of our center over a 9 month period. The main inclusion criteria was natural conception resulting in delivery, whereas ART pregnancies and pregnancies resulting from contraceptive method failure were excluded. 2510 valid interviews were collected. The following parameters were investigated: time to pregnancy (TTP), age and smoking (in both women and men), previous pregnancies and intercourse frequency. A mathematical formula was developed to predict the per-month fecundity rate. Results The cumulative fecundity rate (CFR) was 29.08 %, 54.26%, 68.61%, 89.88%, 96.95% and 98.63% (at 1-3-6-9-12-24-36 months) whereas between the 12th and 36th month the average Per-month fecundity rate (PMFR) ranged between 8.5 and 7.5%. Only 1.68% of pregnancies occurred between the 24th and 36th month, and only 1.37% afterwards. The best fecundity markers were obtained in the group with 7-8 bouts of sexual intercourse per week. The group of women who were < 25 years as well as men who were < 25 years had lower fecundity markers than those between 25 and 40. Conclusions Fertile couples have a not-negligible PMFR between the 12th and the 36th month which should be considered when planning fertility studies. The lower FR observed in women and men < 25 years deserves more study. If the coital frequency is 2-3 per week or more, FR is not impaired, but even better results were obtained with 7-8/week.

Gametogenesis is a complex and sex-specific multistep process during which the gonadal somatic niche plays an essential regulatory role. One of the most crucial steps during human female gametogenesis is the formation of primordial follicles, the functional unit of the ovary that constitutes the pool of follicles available at birth during the entire reproductive life. However, the relation between human fetal germ cells (hFGCs) and gonadal somatic cells during the formation of the primordial follicles remains largely unexplored. We have discovered that hFGCs can form multinucleated syncytia, some connected via interconnecting intercellular bridges, and that not all nuclei in hFGC-syncytia were synchronous regarding meiotic stage. As hFGCs progressed in development, pre-granulosa cells formed protrusions that seemed to progressively constrict individual hFGCs, perhaps contributing to separate them from the multinucleated syncytia. Our findings highlighted the cell-cell interaction and molecular dynamics between hFGCs and (pre)granulosa cells during the formation of primordial follicles in humans. Knowledge on how the pool of primordial follicle is formed is important to understand human infertility.

Background:The purpose of the study was to assess whether the coadministration of 150 IU of recombinant LH instead of 75 IU in women aged 35-39 improves the results in agonist ICSI cycles stimulated with 300 IU of recombinant FSH.Methods:In this study, two ovarian stimulation protocols coexisted which were identical except in the administered dose of recombinant LH, for which some patients received 150 IU (n=231) and some received 75 IU (n=216). Both groups received 300 IU of recombinant FSH. Gonadotropins were reimbursed by the National Health System. Statistical analysis was performed by Student's t test, χ2, and ANCOVA. Significance level was established at p=0.05.Results:The number of retrieved oocytes was slightly higher in the 300/150 group (9.06±5.53 vs. 8.61±5.11), but the differences were not significant. Results were similar with the number of metaphase II oocytes (7.18±4.86 vs. 6.72±4.72) and the number of fertilized oocytes (4.64±3.2 vs. 4.23±2.72). The per-transfer clinical pregnancy rates exhibited close similarity between both groups (32.84% vs. 32.46%), as did the per-transfer live birth rates (29.90% vs. 30.37%) and the implantation rate. The rate of hyperstimulation syndrome (OHSS) as well as the rate of cancellation due to OHHS risk was similar in both groups. There was also no difference in the miscarriage rate. When results were expressed by per started cycle or by oocyte pick-up, the results remained very similar in both groups.Conclusion:In women aged 35-39 undergoing ovarian stimulation with recombinant FSH in agonist cycles, the coadministration of 75 or 150 UI of recombinant LH did not influence pregnancy rates. However, a slight increase in the number of retrieved oocytes should not be disregarded.

Considerable costs are associated with infertility treatment, but little evidence is available on the main drivers of treatment costs. This cost analysis investigated key costs for treatment with assisted reproductive technology (ART) and the proportion of costs attributed to the acquisition of recombinant human follicle-stimulating hormone (r-hFSH) alfa originator for one fresh embryo transfer (ET) leading to a live birth in Spain, Norway, the UK, Germany, Denmark, South Korea, Australia, and New Zealand. The total costs for one ART cycle with a fresh ET leading to a live birth varied between countries (€4108–€12,314). Costs for pregnancy and live birth were the major contributors in European countries, and the costs of oocyte retrieval, monitoring during ovarian stimulation, pregnancy, and live birth were the top contributors in the Asia-Pacific countries, included in this analysis. Acquisition costs for r-hFSH alfa originator contributed to only 5%–17% of the total costs of one ART cycle with one fresh ET leading to a live birth.