Mosaic human preimplantation embryos and their developmental potential in a prospective, non-selection clinical trial.

Chromosome imbalance (aneuploidy) is the major cause of pregnancy loss and congenital disorders in humans. Analyses of small biopsies from human embryos suggest that aneuploidy commonly originates during early divisions, resulting in mosaicism. However, the developmental potential of mosaic embryos remains unclear. We followed the distribution of aneuploid chromosomes across 73 unselected preimplantation embryos and 365 biopsies, sampled from four multifocal trophectoderm (TE) samples and the inner cell mass (ICM). When mosaicism impacted fewer than 50% of cells in one TE biopsy (low-medium mosaicism), only 1% of aneuploidies affected other portions of the embryo. A double-blinded prospective non-selection trial (NCT03673592) showed equivalent live-birth rates and miscarriage rates across 484 euploid, 282 low-grade mosaic, and 131 medium-grade mosaic embryos. No instances of mosaicism or uniparental disomy were detected in the ensuing pregnancies or newborns, and obstetrical and neonatal outcomes were similar between the study groups. Thus, low-medium mosaicism in the trophectoderm mostly arises after TE and ICM differentiation, and such embryos have equivalent developmental potential as fully euploid ones.

COVID-19 and ART: the view of the Italian Society of Fertility and Sterility and Reproductive Medicine.

The COVID-19 pandemic is an unprecedented global situation. As assisted reproductive technology (ART) specialists, we should be cautious, carefully monitoring the situation while contributing by sharing novel evidence to counsel our patients, both pregnant women and would-be mothers. Time to egg collection and drop-out rates are critical parameters for scheduling treatments once the curve of infections has peaked and plateaued in each country. In order to reduce the values for these two parameters, infertile patients now require even more support from their IVF team: urgent oocyte collection for oncology patients must be guaranteed, and oocyte retrievals for women of advanced maternal age and/or reduced ovarian reserve cannot be postponed indefinitely. This document represents the position of the Italian Society of Fertility and Sterility and Reproductive Medicine (SIFES-MR) in outlining ART priorities during and after this emergency.

Male age: negative impact on sperm DNA fragmentation.

The main goal of semen processing in Assisted Reproductive Techniques (ART) is to select sperm with good viability and, at the same time, remove Reactive Oxygen Species (ROS) sources (such as leukocytes) and reduce the percentage of morphologically abnormal sperm for fertility treatment. We performed a comparative analysis on sperm DNA fragmentation after Density Gradient Centrifugation (DGC) using products sold by two competing companies. Our results showed comparable DNA Fragmentation Index (DFI) after treatment with both DGC products. However, in both cases, a comparable number of samples do not benefit from the treatment. Interestingly, increasing evidences indicated that male age has a negative impact on sperm DNA fragmentation, but the mechanisms underlying age-dependent patterns of sperm decline have not yet been fully understood. Thus, we performed a comparative analysis of DFI before and after treatment with DGC products in age-stratified sample populations. Our results showed a worsening of the baseline DFI in the eldest group and the benefits of DGC on sperm DNA were compromised. In conclusion, our work consolidates the current evidences suggesting that both paternal and maternal aging, critically affects reproductive success.

Effect of superoxide dismutase supplementation on sperm DNA fragmentation.

BACKGROUND: antioxidants supplementation improves sperm quality, but few trials have analyzed the effects on sperm DNA fragmentation (SDF). This study compares the effectiveness of SOD-based antioxidant supplementation plus hydroxytyrosol and carnosol in reducing SDF with other antioxidants without SOD, hydroxytyrosol, and carnosol. MATERIALS AND METHODS: men with high SDF at baseline were selected in our clinical database. The patients taken into account had a 2-month control. SDF was measured by Sperm Chromatin Dispersion test (SCD). Untreated men were used as a control group. The remaining subjects received some oral antioxidant supplements (12 different combinations of both hydrophilic and lipophilic antioxidants), with some of them receiving nutritional support with a SOD-based antioxidant supplementation plus hydroxytyrosol and carnosol. RESULTS: 118 men were selected for a retrospective study. Mean age 39.3 ± 5.4 years. Fifteen had no treatment, 55 were treated with a SOD-based antioxidant supplementation plus hydroxytyrosol and carnosol, and 48 took some antioxidant supplements for 2 months. Clinically, variations of at least 10% in baseline values of classic semen parameters and sperm DNA fragmentation were taken into consideration. Classic seminal parameters did not vary significantly in the three groups, with the exception of viability (p = 0.001). We assessed which of the active substances (no. 19) in different formulations were associated with variations in SDF. In the multivariable analysis of the 7 active substances that passed the univariable analysis, only the SOD molecule appeared to be linked to an improvement in SDF (< 0.0001). In detail, only one patient in the control group showed a spontaneous improvement in SDF (6%), compared to 16/48 (33%) of those taking various oral antioxidant supplements, and 31/55 (56%) of those taking a SOD-based antioxidant supplementation plus hydroxytyrosol and carnosol. CONCLUSIONS: SOD-based antioxidant supplementation plus hydroxytyrosol and carnosol seems to provide a better chance of improving sperm DNA integrity than other classical antioxidant molecules.

Preimplantation genetic diagnosis for aneuploidy testing in women older than 44 years: a multicenter experience.

OBJECTIVE: To report laboratory and clinical outcomes in preimplantation genetic diagnosis for aneuploidies (PGD-A) cycles for women 44 to 47 years old. DESIGN: Multicenter, longitudinal, observational study. SETTING: In vitro fertilization (IVF) centers. PATIENT(S): One hundred and thirty-seven women aged 44.7 ± 0.7 years (range: 44.0-46.7) undergoing 150 PGD-A cycles during April 2013 to January 2016. INTERVENTION(S): Quantitative polymerase chain reaction-based PGD-A on trophectoderm biopsies and cryopreserved euploid single-embryo transfer (SET). MAIN OUTCOMES MEASURE(S): Primary outcome measure: delivery rate per cycle; secondary outcome measures: miscarriage rate, and the rate and reasons for cycle cancelation with subanalyses for female age and number of metaphase 2 oocytes retrieved. RESULT(S): In 102 (68.0%) of 150 cycles blastocyst development was obtained, but only 21 (14.0%) were euploid blastocysts. The overall euploidy rate was 11.8% (22 of 187). Twenty-one SET procedures were performed, resulting in 13 clinical pregnancies, of which 1 miscarried and 12 delivered. The delivery rate was 57.1% per transfer, 8.0% per cycle, and 8.8% per patient. The logistic regression analysis found that only female age (odds ratio 0.78) and number of metaphase 2 oocytes retrieved (odds ratio 1.25) statistically significantly correlated with the likelihood of delivery. The delivery rate per cycle was 10.6% (11 of 104) in patients aged 44.0 to 44.9 years and 2.6% in patients aged 45.0 to 45.9 years (n = 1 of 38). No euploid blastocysts were found for patients older than 45.0 years. CONCLUSION(S): Extensive counseling based on biological and clinical data should be provided to women older than 43 years who are requesting IVF because of their very low odds of success and high risk for embryonic aneuploidies. Nevertheless, the low miscarriage and good delivery rates reported in this study in women with good ovarian reserve aged 44 should encourage the use of PGD-A in this population.

Treatment with human, recombinant FSH improves sperm DNA fragmentation in idiopathic infertile men depending on the FSH receptor polymorphism p.N680S: a pharmacogenetic study.

STUDY QUESTION: Does the sperm DNA fragmentation index (DFI) improve depending on the FSH receptor (FSHR) genotype as assessed by the nonsynonymous polymorphisms rs6166 (p.N680S) after 3 months of recombinant FSH treatment in men with idiopathic infertility? SUMMARY ANSWER: FSH treatment significantly improves sperm DFI only in idiopathic infertile men with the p.N680S homozygous N FSHR. WHAT IS KNOWN ALREADY: FSH, fundamental for spermatogenesis, is empirically used to treat male idiopathic infertility and several studies suggest that DFI could be a candidate predictor of response to FSH treatment, in terms of probability to conceive. Furthermore, it is known that the FSHR single nucleotide polymorphism (SNP) rs6166 (p.N680S) influences ovarian response in women and testicular volume in men. STUDY DESIGN, SIZE AND DURATION: A multicenter, longitudinal, prospective, open-label, two-arm clinical trial was performed. Subjects enrolled were idiopathic infertile men who received 150 IU recombinant human FSH s.c. every other day for 12 weeks and were followed-up for a further 12 weeks after FSH withdrawal. Patients were evaluated at baseline, at the end of treatment and at the end of follow-up. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eighty-nine men with idiopathic infertility carrier of the FSHR p.N680S homozygous N or S genotype, FSH ≤ 8 IU/l and DFI >15%, were enrolled. A total of 66 patients had DFI analysis completed on at least two visits. DFI was evaluated in one laboratory by TUNEL/PI (propidium iodide) assay coupled to flow cytometry, resolving two different fractions of sperm, namely the 'brighter' and 'dimmer' sperm DFI fractions. MAIN RESULTS AND THE ROLE OF CHANCE: Thirty-eight men (57.6%) were carriers of the p.N680S homozygous N and 28 (42.4%) of the homozygous S FSHR. Sperm concentration/number was highly heterogeneous and both groups included men ranging from severe oligozoospermia to normozoospermia. Total DFI was significantly lower at the end of the study in homozygous carriers of the p.N680S N versus p.N680S S allele (P = 0.008). Total DFI decreased significantly from baseline to the end of the study (P = 0.021) only in carriers of the p.N680S homozygous N polymorphism, and this decrease involved the sperm population containing vital sperm (i.e. brighter sperm) (P = 0.008). The dimmer sperm DFI fraction, including only nonvital sperm, was significantly larger in p.N680S S homozygous patients than in homozygous N men (P = 0.018). Total DFI was inversely related to total sperm number (P = 0.020) and progressive sperm motility (P = 0.014). When patients were further stratified according to sperm concentration (normoozospermic versus oligozoospermic) or -211G>T polymorphism in the FSHB gene (rs10835638) (homozygous G versus others), the significant improvement of sperm DFI in FSHR p.N680S homozygous N men was independent of sperm concentration and associated with the homozygous FSHB -211G>T homozygous G genotype. LIMITATIONS, REASONS FOR CAUTION: The statistical power of the study is 86.9% with alpha error 0.05. This is the first pharmacogenetic study suggesting that FSH treatment induces a significant improvement of total DFI in men carriers of the p.N680S homozygous N FSHR; however, the results need to be confirmed in larger studies using a personalized FSH dosage and treatment duration. WIDER IMPLICATIONS OF THE FINDINGS: The evaluation of sperm DFI as a surrogate marker of sperm quality, and of the FSHR SNP rs6166 (p.N680S), might be useful to predict the response to FSH treatment in men with idiopathic infertility. STUDY FUNDING/COMPETING INTERESTS: The study was supported by an unrestricted grant to M.S. and H.M.B. from Merck Serono that provided the drug used in the study. MS received additional grants from Merck Serono and IBSA as well as honoraria from Merck Serono. The remaining authors declare that no conflicts of interest are present. TRIAL REGISTRATION NUMBER: EudraCT number 2010-020240-35.

How to define, diagnose and treat poor responders? Responses from a worldwide survey of IVF clinics.

Poor responders represent a significant percentage of couples treated in IVF units (10-24%), but the standard definition of poor responders remains uncertain and consequently optimal treatment options remain subjective and not evidence-based. In an attempt to provide uniformity on the definition, diagnosis and treatment of poor responders, a worldwide survey was conducted asking IVF professionals a set of questions on this complex topic. The survey was posted on www.IVF-worldwide.com, the largest and most comprehensive IVF-focused website for physicians and embryologists. A total of 196 centres replied, forming a panel of IVF units with a median of 400 cycles per year. The present study shows that the definition of poor responders is still subjective, and many practices do not use evidence-based treatment for this category of patients. Our hope is that by leveraging the great potential of the internet, future studies may provide immediate large-scale sampling to standardize both poor responder definition and treatment options.

Clomiphene citrate versus high doses of gonadotropins for in vitro fertilisation in women with compromised ovarian reserve: a randomised controlled non-inferiority trial.

BACKGROUND: The aim of the present randomised controlled non-inferiority trial is to test whether in women with compromised ovarian reserve requiring in vitro fertilisation, a protocol of ovarian stimulation using exclusively clomiphene citrate performs similarly to a regimen with high doses of gonadotropins. METHODS: Women with day 3 serum FSH > 12 IU/ml on at least two occasions or previous poor response to hyper-stimulation were recruited at four Italian infertility units. Selected women were allocated to clomiphene citrate 150 mg/day from day 3 to day 7 of the cycle (n=145) or to a short protocol with GnRH agonist 0.1 mg and recombinant FSH 450 IU daily (n=146). They were randomised by means of a computer-generated list into two groups. The study was not blinded. The main outcome of the study was the delivery rate per started cycle. RESULTS: The study was interrupted after the scheduled two years of recruitment before reaching the sample size. 148 women were allocated to clomiphene citrate and 156 to the short protocol with high doses of gonadotropins; 124 and 125 participants were analysed in the groups, respectively. Women allocated to high doses of gonadotropins retrieved more oocytes and had a higher probability to perform embryo-transfer. However, the chances of success were similar. The delivery rate per started cycle in women receiving clomiphene citrate and high-dose gonadotropins was 3% (n=5) and 5% (n=7), respectively (p=0.77). The mean estimated cost per delivery in the two groups was 81,294 and 113,107 Euros, respectively. No side-effects or adverse events were observed. CONCLUSIONS: In women with compromised ovarian reserve selected for in vitro fertilisation, ovarian stimulation with clomiphene citrate or high-dose gonadotropins led to similar chances of pregnancy but the former is less expensive. TRIAL REGISTRATION: Trial registered on http://www.clinicaltrials.gov (NCT01389713).

Sperm protein 17 is expressed in the sperm fibrous sheath.

BACKGROUND: Sperm protein 17 (Sp17) is a highly conserved mammalian protein characterized in rabbit, mouse, monkey, baboon, macaque, human testis and spermatozoa. mRNA encoding Sp17 has been detected in a range of murine and human somatic tissues. It was also recognized in two myeloma cell lines and in neoplastic cells from patients with multiple myeloma and ovarian carcinoma. These data all indicate that Sp17 is widely distributed in humans, expressed not only in germinal cells and in a variety of somatic tissues, but also in neoplastic cells of unrelated origin. METHODS: Sp17 expression was analyzed by immunocytochemistry and transmission electron microscopy on spermatozoa. RESULTS: Here, we demonstrate the ultrastructural localization of human Sp17 throughout the spermatozoa flagellar fibrous sheath, and its presence in spermatozoa during in vitro states from their ejaculation to the oocyte fertilization. CONCLUSION: These findings suggest a possible role of Sp17 in regulating sperm maturation, capacitation, acrosomal reaction and interactions with the oocyte zona pellucida during the fertilization process. Further, the high degree of sequence conservation throughout its N-terminal half, and the presence of an A-kinase anchoring protein (AKAP)-binding motif within this region, suggest that Sp17 might play a regulatory role in a protein kinase A-independent AKAP complex in both germinal and somatic cells.

In vitro maturation of human oocytes in a follicle-mimicking three-dimensional coculture.

OBJECTIVE: To verify the hypothesis that a three-dimensional, follicle-mimicking structure enhances in vitro maturation yields without hormonal supplementation in an in vitro maturation program. DESIGN: Feasibility study; 204 anonymous denuded germinal vesicles retrieved from gonadotropin-treated women were cultured for 48 hours without hormonal supplementation in microdrop culture or in a three-dimensional coculture with granulosa cells in a barium alginate membrane. SETTING: An assisted reproduction center in Italy. PATIENT(S): One hundred twenty-two informed women. INTERVENTION(S): Germinal vesicles retrieved after ovarian stimulation were denuded and cultured without hormonal supplementation in microdrop culture or in a three-dimensional coculture with granulosa cells enclosed in the core of a barium alginate capsule. MAIN OUTCOME MEASURE(S): Oocyte maturation and morphological investigation of follicle-mimicking structures. RESULT(S): Statistically significantly higher oocyte maturation yields were obtained by using the three-dimensional coculture system enclosed in a barium alginate membrane (after 48 hours: coculture, 90.3%; microdrop, 52.0%; odds ratio, 8.51). Morphological investigation indicated that after 48 hours of coculture, the decumulated oocyte appeared to be surrounded by a pseudocumulus structure that lies on the inner surface of the alginate membrane and protrudes toward the capsule's lumen. CONCLUSION(S): The maturation yield of cocultured oocytes is eightfold higher than that achieved with a microdrop maturation technique, making a higher number of gametes available for IVF programs. Stereomicroscopic morphological survey indicates a reassembly of granulosa cells surrounding oocyte zona, mimicking an antral cumulus oophorus.

Recombinant gonadotrophins associated with GnRH antagonist (cetrorelix) in ovarian stimulation for ICSI: comparison of r-FSH alone and in combination with r-LH.

OBJECTIVE: The objective was to verify the outcome of intracytoplasmic sperm injection (ICSI) with ovulation induction performed with GnRH antagonists, comparing the use of recombinant follicle-stimulating hormone (r-FSH) alone and in combination with recombinant luteinizing hormone (r-LH) in a prospective and randomized trial. STUDY DESIGN: Forty male-factor infertile normo-ovulatory patients undergoing ovarian stimulation for ICSI took part in the study. After initiating ovarian stimulation with only r-FSH, all patients were treated with GnRH antagonist (cetrorelix). When beginning cetrorelix administration, the patients were randomized into two groups: in group I, 20 patients continued to receive r-FSH alone and in group II, 20 patients received combined r-FSH and r-LH. The number of metaphase II oocytes, estradiol concentration at the time of hCG administration, fertilization rate, grade 1 embryo rate, pregnancy rate per cycle, and implantation rate were measured. Results are expressed as mean+/-S.D. RESULTS: In group I, the women's age was 32.3+/-2.30 years, and FSH concentration was 7.8+/-1.7 IU/ml. In group II, the women's age was 32.2+/-2.46 years and FSH concentration was 7.5+/-1.7 IU/ml. The number of oocytes retrieved was 9.6+/-2.9 and the number of metaphase II oocytes was 6.7+/-2.2 in group I. In group II the number of retrieved oocytes were 9.9+/-2.6 and the number of metaphase II oocytes 6.9+/-2.1 (p>0.05). Estradiol concentration at the time of hCG was 4.6+/-1.8 nm/l in group I and 6.7+/-2.0 nm/l in group II (p<0.01). Fertilization rate was 73.0% in group I versus 78.2% in group II. In group I, we obtained 53.9% of grade 1 embryos versus 54.4% in group II (p>0.05). Pregnancy and implantation rates in group I were 30.0 and 16.7%, respectively and in group II 35.0 and 20.4%, respectively (p>0.05). CONCLUSIONS: The use of recombinant LH in addition to recombinant FSH may prevent a decrease in estradiol after GnRH antagonist administration, but does not influence positively the outcome of oocyte number, maturation, embryo quality, fertilization rate, pregnancy rate per cycle, and implantation rate.

Associação de FSH e LH recombinantes na estimulação ovariana / Association of recombinant FSH and LH in ovarian stimulation

Os autores revisam a fisiologia do ciclo ovulatório e o papel das gonadotropinas na indução da ovulação, em pacientes com distúrbios ovulatórios e no desenvolvimento folicular múltiplo para técnicas de reprodução assistida. O emprego de gonadotropinas com atividade de LH e o uso do LH recombinante associado ao FSH são discutidos. Os autores reafirmam que a administração de gonadotropinas com atividade de LH é essencial na ovulação por hipogonadismo hipogonadotrópico. O uso do LH recombinante associado ao FSH pode ser particularmente benéfico nos regimes de estimulação ovariana com antagonistas do GnRH, prevenindo a queda nos níveis de estradiol e diminuindo as necessidades de FSH

FSH and LH together in ovarian stimulation.

The authors review the physiology of the ovulatory cycle and the role of the gonadotrophins in ovulation induction in patients with anovulatory disorders and in multifollicular development for assisted reproductive technologies. The use of gonadotrophins with luteinizing hormone (LH) activity and the use of recombinant LH associated with follicle stimulating hormone (FSH) are discussed. The authors point out that administration of gonadotrophins with LH activity is essential in hypogonadotropic hypogonadal anovulation, and data available in the medical literature allow the conclusion that recombinant LH may be added to all ovarian stimulation protocols because it is difficult to determine which patients will benefit from LH administration and there is no evidence that LH affects adversely the outcome of ovarian stimulation. The use of recombinant LH in addition to recombinant FSH may be particularly useful when a GnRH antagonist is associated with the ovarian stimulation regimen, by preventing the fall in estradiol and diminishing FSH requirements.