Role of thyroid stimulating hormone in the maintenance and functioning of the human corpus luteum.

PURPOSE: To evaluate the impact of high thyroid stimulating hormone (TSH) levels on human granulosa-luteal (hGL) cells. METHODS: hGL cells were isolated from follicular aspirates derived from patients undergoing IVF treatment without any thyroid disorder (serum TSH 0.5-2 mU/L). Cells were cultured at 37 °C in DMEM, supplemented with 5% FBS. The cells were treated with 1 nM LH and increasing concentrations of TSH. At the end of culture, conditioned medium and cells were collected to analyze progesterone production, cell viability, and mRNA levels of genes involved in the steroidogenesis process. Human ovarian tissues were analyzed for TSH receptor (TSHR) expression by IHC. RESULTS: The expression of TSHR was detected in human corpus luteum by IHC and in hGL by RT-PCR. In hGL cells, TSH treatment did not modulate progesterone production nor the expression of steroidogenic genes, such as p450scc and HSD3b 1/2. However, TSH induced a dose-dependent increase in cell death. Finally, TSH did not affect LH-induced p450scc and HSD3b1/2 expression while LH partially reverted TSH negative effect on cell death in hGL. CONCLUSIONS: Elevated TSH levels in hypothyroid women may be associated with impaired CL functioning and maintenance. These findings open a new line of research for the importance of the treatment of women with thyroid dysfunction that could contribute to the onset of infertility.

Improved clinical utility of preimplantation genetic testing through the integration of ploidy and common pathogenic microdeletions analyses.

STUDY QUESTION: Can chromosomal abnormalities beyond copy-number aneuploidies (i.e. ploidy level and microdeletions (MDs)) be detected using a preimplantation genetic testing (PGT) platform? SUMMARY ANSWER: The proposed integrated approach accurately assesses ploidy level and the most common pathogenic microdeletions causative of genomic disorders, expanding the clinical utility of PGT. WHAT IS KNOWN ALREADY: Standard methodologies employed in preimplantation genetic testing for aneuploidy (PGT-A) identify chromosomal aneuploidies but cannot determine ploidy level nor the presence of recurrent pathogenic MDs responsible for genomic disorders. Transferring embryos carrying these abnormalities can result in miscarriage, molar pregnancy, and intellectual disabilities and developmental delay in offspring. The development of a testing strategy that integrates their assessment can resolve current limitations and add valuable information regarding the genetic constitution of embryos, which is not evaluated in PGT providing new level of clinical utility and valuable knowledge for further understanding of the genomic causes of implantation failure and early pregnancy loss. To the best of our knowledge, MDs have never been studied in preimplantation human embryos up to date. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort analysis including blastocyst biopsies collected between February 2018 and November 2021 at multiple collaborating IVF clinics from prospective parents of European ancestry below the age of 45, using autologous gametes and undergoing ICSI for all oocytes. Ploidy level determination was validated using 164 embryonic samples of known ploidy status (147 diploids, 9 triploids, and 8 haploids). Detection of nine common MD syndromes (-4p=Wolf-Hirschhorn, -8q=Langer-Giedion, -1p=1p36 deletion, -22q=DiGeorge, -5p=Cri-du-Chat, -15q=Prader-Willi/Angelman, -11q=Jacobsen, -17p=Smith-Magenis) was developed and tested using 28 positive controls and 97 negative controls. Later, the methodology was blindly applied in the analysis of: (i) 100 two pronuclei (2PN)-derived blastocysts that were previously defined as uniformly euploid by standard PGT-A; (ii) 99 euploid embryos whose transfer resulted in pregnancy loss. PARTICIPANTS/MATERIALS, SETTING, METHODS: The methodology is based on targeted next-generation sequencing of selected polymorphisms across the genome and enriched within critical regions of included MD syndromes. Sequencing data (i.e. allelic frequencies) were analyzed by a probabilistic model which estimated the likelihood of ploidy level and MD presence, accounting for both sequencing noise and population genetics patterns (i.e. linkage disequilibrium, LD, correlations) observed in 2504 whole-genome sequencing data from the 1000 Genome Project database. Analysis of phased parental haplotypes obtained by single-nucleotide polymorphism (SNP)-array genotyping was performed to confirm the presence of MD. MAIN RESULTS AND THE ROLE OF CHANCE: In the analytical validation phase, this strategy showed extremely high accuracy both in ploidy classification (100%, CI: 98.1-100%) and in the identification of six out of eight MDs (99.2%, CI: 98.5-99.8%). To improve MD detection based on loss of heterozygosity (LOH), common haploblocks were analyzed based on haplotype frequency and LOH occurrence in a reference population, thus developing two further mathematical models. As a result, chr1p36 and chr4p16.3 regions were excluded from MD identification due to their poor reliability, whilst a clinical workflow which incorporated parental DNA information was developed to enhance the identification of MDs. During the clinical application phase, one case of triploidy was detected among 2PN-derived blastocysts (i) and one pathogenic MD (-22q11.21) was retrospectively identified among the biopsy specimens of transferred embryos that resulted in miscarriage (ii). For the latter case, family-based analysis revealed the same MD in different sibling embryos (n = 2/5) from non-carrier parents, suggesting the presence of germline mosaicism in the female partner. When embryos are selected for transfer based on their genetic constitution, this strategy can identify embryos with ploidy abnormalities and/or MDs beyond aneuploidies, with an estimated incidence of 1.5% (n = 3/202, 95% CI: 0.5-4.5%) among euploid embryos. LIMITATIONS, REASONS FOR CAUTION: Epidemiological studies will be required to accurately assess the incidence of ploidy alterations and MDs in preimplantation embryos and particularly in euploid miscarriages. Despite the high accuracy of the assay developed, the use of parental DNA to support diagnostic calling can further increase the precision of the assay. WIDER IMPLICATIONS OF THE FINDINGS: This novel assay significantly expands the clinical utility of PGT-A by integrating the most common pathogenic MDs (both de novo and inherited ones) responsible for genomic disorders, which are usually evaluated at a later stage through invasive prenatal testing. From a basic research standpoint, this approach will help to elucidate fundamental biological and clinical questions related to the genetics of implantation failure and pregnancy loss of otherwise euploid embryos. STUDY FUNDING/COMPETING INTEREST(S): No external funding was used for this study. S.C., M.F., F.C., P.Z., I.P., L.G., C.P., M.P., D.B., J.J.-A., D.B.-J., J.M.-V., and C.R. are employees of Igenomix and C.S. is the head of the scientific board of Igenomix. A.C. and L.P. are employees of JUNO GENETICS. Igenomix and JUNO GENETICS are companies providing reproductive genetic services. TRIAL REGISTRATION NUMBER: N/A.

Clinical validity and utility of preconception expanded carrier screening for the management of reproductive genetic risk in IVF and general population.

STUDY QUESTION: What is the clinical validity and utility of preconception Expanded Carrier Screening (ECS) application on the management of prospective parents? SUMMARY ANSWER: The high detection rate of at-risk couples (ARCs) and the high proportion opting for IVF/preimplantation genetic testing (PGT) treatment demonstrate the clinical utility of ECS in the preconception space in IVF and general population. WHAT IS KNOWN ALREADY: About 2-4% of couples are at risk of conceiving a child with an autosomal recessive or X-linked genetic disorder. In recent years, the increasing cost-effectiveness of genetic diagnostic techniques has allowed the creation of ECS panels for the simultaneous detection of multiple recessive disorders. Comprehensive preconception genetic screening holds the potential to significantly improve couple's genetic risk assessment and reproductive planning to avoid detectable inheritable genetic offspring. STUDY DESIGN, SIZE, DURATION: A total of 3877 individuals without a family history of genetic conditions were analyzed between January 2017 and January 2020. Of the enrolled individuals, 1212 were gamete donors and 2665 were patients planning on conceiving from both the IVF and the natural conception group. From the non-donor cohort, 1133 were analyzed as individual patients, while the remaining ones were analyzed as couples, for a total of 766 couples. PARTICIPANTS/MATERIALS, SETTING, METHODS: A focused ECS panel was developed following American College of Obstetrics and Gynecology ACOG-recommended criteria (prevalence, carrier rate, severity), including highly penetrant severe childhood conditions. Couples were defined at-risk when both partners carried an autosomal recessive pathogenic/likely pathogenic variant (PLP) on the same gene or when the woman was a carrier of an X-linked PLP variant. ARC detection rate defined the clinical validity of the ECS approach. Clinical utility was evaluated by monitoring ARCs reproductive decision making. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 402 individuals (10.4%) showed PLP for at least one of the genes tested. Among the 766 couples tested, 173 showed one carrier partner (22.6%), whereas 20 couples (2.6%) were found to be at increased risk. Interestingly, one ARC was identified as a result of cascade testing in the extended family of an individual carrying a pathogenic variant on the Survival Of Motor Neuron 1SMN1 gene. Of the identified ARCs, 5 (0.7%) were at risk for cystic fibrosis, 5 (0.7%) for fragile X syndrome, 4 (0.5%) for spinal muscular atrophy, 4 (0.5%) for Beta-Thalassemia/Sickle Cell Anemia, 1 (0.1%) for Smith-Lemli-Opitz Syndrome and 1 (0.1%) for Duchenne/Becker Dystrophy. Fifteen ARCs were successfully followed up from both the IVF and the natural conception groups. All of these (15/15) modified their reproductive planning by undergoing ART with Preimplantation Genetic Testing for Monogenic disease and Aneuploidies (PGT-M and PGT-A). To date, 6/15 (40%) couples completed their PGT cycle with euploid/unaffected embryos achieving a pregnancy after embryo transfer and three of them have already had an unaffected baby. LIMITATIONS, REASONS FOR CAUTION: The use of a limited panel of core gene-disease pairs represents a limitation on the research perspective as it can underestimate the rate of detectable carriers and ARCs in this cohort of prospective parents. Expanding the scope of ECS to a larger panel of conditions is becoming increasingly feasible, thanks to a persistent technological evolution and progressive cataloging of gene-disease associations. WIDER IMPLICATIONS OF THE FINDINGS: These results highlight the potential clinical validity and utility of ECS in reducing the risk of a pregnancy affected by a detectable inheritable genetic condition. The steady reduction in the costs of genetic analyses enables the expansion of monogenic testing/screening applications at the preimplantation stage, thus, providing valid decisional support and reproductive autonomy to patients, particularly in the context of IVF. STUDY FUNDING/COMPETING INTEREST(S): No external funding was used for this study. A.C., M.F., S.C., M.P., L.G., and C.P. are employees of Igenomix Italy. C.S. is the head of the scientific board of Igenomix. TRIAL REGISTRATION NUMBER: N/A.

Continuous embryo culture elicits higher blastulation but similar cumulative delivery rates than sequential: a large prospective study.

PURPOSE: To assess whether continuous embryo culture involves better embryological and/or clinical outcomes than sequential. METHODS: Prospective study at a private IVF center. All consecutive IVF cycles (September 2013-2015) fulfilling the inclusion criteria underwent embryo culture in either Continuous-Single-Culture-Media (CSCM, n = 972) or sequential media (Quinn's Advantage, n = 514), respectively. ICSI, blastocyst culture in either standard (MINC) or undisturbed (Embryoscope) incubation, transfer (until September 2016), and pregnancy follow-up (until September 2017) were performed. When aneuploidy testing was required, trophectoderm biopsy and qPCR were performed. Sub-analyses and logistic regression corrected for confounders were performed. The primary outcomes were overall blastocyst rate per oocyte and mean blastocyst rate per cycle. The sample size was defined to reach 95 and 80% statistical power for the former and the latter outcome, respectively. Secondary outcomes were euploidy (if assessed), cumulative delivery rates, gestational age, and birthweight. RESULTS: Continuous embryo culture resulted into a higher overall blastocyst rate per inseminated oocyte than sequential (n = 2211/5841, 37.9% vs. 1073/3216, 33.4%; p < 0.01), confirmed also from a cycle-based analysis (mean blastocyst rate: 38.7% ± 29.7% vs. 34.3% ± 29.4%; p = 0.01). The continuous media (OR = 1.23), the undisturbed incubation system (OR = 1.22), the maternal age (OR = 0.92), and the sperm factor (OR = 0.85) were outlined as positive predictors of blastulation. However, the cumulative delivery rates per ended cycle (i.e., delivery achieved or no blastocyst produced or left; > 90%) were comparable in the two groups (n = 244/903, 27.0% vs. 129/475, 27.2%). The neonatal outcomes were similar. CONCLUSIONS: Continuous culture involves better embryological but similar clinical outcomes than sequential. This large prospective study supports the absence of clinical disparity among the two approaches.

No evidence of association between blastocyst aneuploidy and morphokinetic assessment in a selected population of poor-prognosis patients: a longitudinal cohort study.

Recent studies involving a limited number of patients have indicated a correlation between aneuploidy and various morphokinetic parameters during preimplantation development. The results among different groups, however, have been inconsistent in identifying the parameters that are able to predict chromosomal abnormalities. The aim of this study was to investigate whether aneuploidy of human blastocysts was detectable by specific morphokinetic parameters in patients at increased risk of aneuploidy because of advanced maternal age, history of unsuccessful IVF treatments, or both. A longitudinal cohort study was conducted using 455 blastocysts from 138 patients. Morphokinetic features of preimplantation development were detected in a timelapse incubator. Blastocysts were subjected to trophectodermal biopsy and comprehensive chromosomal screening. Analyses were conducted by means of logistic mixed-effects models, with a subject-specific intercept. No statistical correlation between 16 commonly detected morphokinetic characteristics of in-vitro embryo development and aneuploidy was found. Results suggest that morphokinetic characteristics cannot be used to select euploid blastocysts in poor-prognosis patients regarded as candidates for pre-implantation genetic screening.

PGD for reciprocal and Robertsonian translocations using array comparative genomic hybridization.

BACKGROUND: Fluorescence in situ hybridization (FISH) is the most widely used method for detecting unbalanced chromosome rearrangements in preimplantation embryos but it is known to have several technical limitations. We describe the clinical application of a molecular-based assay, array comparative genomic hybridization (array-CGH), to simultaneously screen for unbalanced translocation derivatives and aneuploidy of all 24 chromosomes. METHODS: Cell biopsy was carried out on cleavage-stage embryos (Day 3). Single cells were first lysed and DNA amplified by whole-genome amplification (WGA). WGA products were then processed by array-CGH using 24sure + arrays, BlueGnome. Balanced/normal euploid embryos were then selected for transfer on Day 5 of the same cycle. RESULTS: Twenty-eight consecutive cycles of preimplantation genetic diagnosis were carried out for 24 couples carrying 18 different balanced translocations. Overall, 187/200 (93.5%) embryos were successfully diagnosed. Embryos suitable for transfer were identified in 17 cycles (60.7%), with transfer of 22 embryos (mean 1.3 ± 0.5). Twelve couples achieved a clinical pregnancy (70.6% per embryo transfer), with a total of 14 embryos implanted (63.6% per transferred embryo). Three patients delivered three healthy babies, during writing, the other pregnancies (two twins and seven singletons) are ongoing beyond 20 weeks of gestation. CONCLUSIONS: The data obtained demonstrate that array-CGH can detect chromosome imbalances in embryos, also providing the added benefit of simultaneous aneuploidy screening of all 24 chromosomes. Array-CGH has the potential to overcome several inherent limitations of FISH-based tests, providing improvements in terms of test performance, automation, sensitivity and reliability.

Hopes and facts about mild ovarian stimulation.

Over the last two decades, easier and less expensive stimulation treatments have been largely replaced by more complex and more demanding protocols. Since the mid-nineties, long-term gonadotrophin-releasing hormone agonist stimulation protocols have been widely used. Such lengthy expensive regimens are not free from short- and long-term risks and complications. Mild stimulation protocols reduce the mean number of days of stimulation, the total amount of gonadotrophins used and the mean number of oocytes retrieved. The proportion of high quality and euploid embryos seems to be higher compared with conventional stimulation protocols and the pregnancy rate per embryo transfer is comparable. Moreover, the reduced costs, the better tolerability for patients and the less time needed to complete an IVF cycle make mild approaches clinically and cost-effective over a given period of time. However, further prospective randomized studies are needed to compare cumulative pregnancy rates between the two protocols. Natural cycle IVF, with minimal stimulation, has been recently proposed as an alternative to conventional stimulation protocols in normo- and poor responder patients. Although acceptable results have been reported, further large prospective randomized studies are needed to better evaluate the efficacy of these minimal regimens compared with conventional stimulation approaches.

GnRH antagonists in ovarian stimulation for ICSI with oocyte restriction: a matched, controlled study.

Italian legislation regarding reproductive medicine limits the number of embryos transferred per attempt to three. Thus, in order to achieve pregnancy, more IVF cycles may be required, generating a need for methods of ovarian stimulation with fewer side effects. The gonadotrophin-releasing hormone (GnRH) antagonists have several advantages in this respect, but there is a debate regarding a possible lower pregnancy rate from resulting cycles. This study evaluated the clinical applicability of GnRH antagonists for ovarian stimulation in young women undergoing intracytoplasmic sperm injection (ICSI) in which only three oocytes can be fertilized. The 200 women treated with GnRH antagonist had a significantly shorter stimulation and lower gonadotrophin consumption, oestradiol concentration, total and mature oocyte recovery as compared with 200 matched controls treated with GnRH agonist. No differences were found between the groups in the number of normal zygotes, total cleaved, transferred and high quality embryos, or in the clinical outcomes. Thus, the previously reported lower pregnancy rate in GnRH antagonist cycles may be related to the oocyte characteristics. Finally, under conditions of oocyte number restriction, the GnRH antagonist-based cycles may be proposed as an efficacious, safe and minimally invasive alternative to GnRH agonist in a standard long protocol.

Management of poor responders in IVF.

Correct controlled ovarian stimulation is of paramount importance in assisted reproductive technologies. Therefore, analysis of the ovarian reserve of the patient is mandatory to tailor the best ovarian stimulation regimen. When the ovarian reserve is reduced, the induction of a multifollicular growth remains a challenge. Several factors could be associated with reduced ovarian response. However, reduced ovarian reserve either in older patients or in young patients represents the most frequent aetiological factor. Whatever is the aetiology, one of the main problems is how to predict a reduced ovarian response, and although several tests have been suggested, no very accurate predictive test is available. A variety of different stimulation protocols have been suggested but the lack of any large-scale, prospective, randomized, controlled trials of the different management strategies and the lack of a uniform definition of the population may result in comparisons of heterogeneous groups of patients, making it difficult to draw any definitive conclusions. Natural cycle IVF may represent an easy and cheap approach in the management of this group of patients. Although no controlled large prospective randomized studies are available to compare the natural IVF procedure with ovarian stimulation IVF in poor responder patients, the efficacy of natural cycle IVF is hampered by high cancellation rates mainly due to untimely LH surge. The use of gonadotrophin-releasing hormone antagonists in the late follicular phase, which reduces the premature LH rise rate, and the improvements in laboratory conditions and fertilization techniques, increase the embryo transfer rates, making this procedure more cost-effective.

Birth of a healthy female after preimplantation genetic diagnosis for Charcot-Marie-Tooth type X.

The X-linked dominant form of Charcot-Marie-Tooth syndrome (CMTX) is a clinically and genetically heterogeneous hereditary disorder of the peripheral nerves caused by mutations in the GJB1 gene that encodes a gap junction protein named connexin 32 (Cx32). Clinically, CMTX is characterized by peripheral motor and sensory deficit with muscle atrophy. A couple with a previous history of pregnancy termination after being diagnosed positive for CMTX by chorionic villus sampling, was referred for preimplantation genetic diagnosis (PGD). The female partner carried the causative H94Q, characterized by a C-->G substitution in codon 94 of exon 2 of the GJB1 gene. Embryos obtained after intracytoplasmic sperm injection (ICSI) were evaluated for the presence of the mother's mutation using polymerase chain reaction (PCR), followed by mutation analysis performed using the minisequencing method. Amelogenin sequences on the X and Y chromosomes were also co-amplified to provide a correlation between embryo gender and mutation presence. A single PGD cycle was performed, involving nine fertilized oocytes, five of which developed into good quality embryos useful for biopsy. Two unaffected embryos were transferred, resulting in a singleton pregnancy followed by the birth of a healthy female.

Zona pellucida solubility and cortical granule complements in human oocytes following assisted reproductive techniques.

In this study the solubility to alpha-chymotrypsin of the zona pellucida (ZP) of human oocytes and polyploid embryos obtained during various clinical procedures of assisted fertilisation (IVF, ICSI, cyropreservation) was evaluated. The aim of the study was to determine whether changes in ZP solubility occur during such procedures and whether abnormal solubility could be likened to fertilisation failure. Correlation between ZP solubility and cortical granule (CG) density was also studied. The results showed that ZP solubility varied considerably among germinal vesicle or metaphase oocytes obtained from different subjects, but was essentially identical for the oocyte cohort obtained from individual women. On the basis of ZP solubility metaphase oocytes were subdivided into two classes: class I, average ZP dissolution time +/- SE = 24.1+/-0.9 min, n = 28; and class II, 46.7+/-2.0 min, n = 13. Prolonged ZP dissolution times of metaphase oocytes were significantly correlated with a low in vitro fertilisation rate in sibling oocytes. The zonae of fertilised eggs (polyploid embryos) showed long solubilisation times (IVF: 45.3+/-3.4 min, n = 18; ICSI: 48.9+/-2.7 min, n = 19). ZP solubility of oocytes that failed to fertilise was intermediate between that of class I metaphase oocytes and embryos (unfertilised IVF: 33.0+/-2.7 min, n = 13; unfertilised ICSI: 43.0+/-2.4 min, n = 9). A moderate spontaneous ZP hardening occurred when metaphase oocytes were cultured for 24 h. Finally, cryopreservation of unfertilised oocytes caused hardening of their ZP, with dissolution times that were comparable to those found in fertilised eggs (49.5+/-2.3 min, n = 10). In most cases, an inverse correlation was found between ZP dissolution time and CG density (longer solubilisation times corresponding to lower CG density). ZP hardening caused by cryopreservation, however, was not associated with a significant reduction in CG density in most of the oocytes examined.

Criteria predicting the absence of spermatozoa in the Sertoli cell-only syndrome can be used to improve success rates of sperm retrieval.

In patients with non-obstructive azoospermia, testicular sperm extraction (TESE) is a method of choice to recover spermatozoa as a male therapeutic approach in intracytoplasmic sperm injection (ICSI) programmes. However, the efficacy of TESE in this indication is burdened by a frequent failure of sperm recovery, which renders useless both the invasive testicular intervention and ovarian stimulation of the patient's spouse. One of the most frequent pathological pictures characterizing complete absence of spermatozoa is germinal aplasia (Sertoli cell- only syndrome or SCOS). Two different histological patterns of SCOS have been already described during the past five decades. These two patterns can be characterized as the congenital (pure) and the secondary (mixed) forms. Both patterns, with different prognosis to retrieve spermatozoa by therapeutic testicular biopsy, are frequently confused when TESE is performed during ICSI programmes. Useful criteria to predict the absence of spermatozoa can be obtained by a definite recognition of the two typical histological patterns during the diagnostic testicular biopsy. The diagnosis of congenital or acquired SCOS can be refined by endocrine, chemical, immunohistochemical and molecular biology aids. Reduction of both sperm retrieval failure and unnecessary ovarian stimulation can be achieved by combination of these methods.

Differentiation of spermatogenic cells during in-vitro culture of testicular biopsy samples from patients with obstructive azoospermia: effect of recombinant follicle stimulating hormone.

In-vitro differentiation of spermatogenic cells is a potential approach to the treatment of male sterility due to spermatogenic arrest. This is a pilot study evaluating meiotic, morphogenetic and cytoplasmic maturation of spermatogenic cells from 18 patients with obstructive azoospermia, during in-vitro culture of partly disintegrated testicular biopsy samples in the presence or absence of recombinant follicle stimulating hormone (rFSH). Meiotic progression was detectable only in the presence of rFSH in culture medium. FSH-dependent condensation, peripheral migration and protrusion of spermatid nuclei, together with FSH-independent flagellar growth, were the main events indicating post-meiotic sperm cell differentiation. rFSH also promoted the progression of spermatid cytoplasmic maturation, reflected by acceleration of acrosomal development. These differentiation events appeared to be mediated by humoral activity of Sertoli cells, without the need for a direct Sertoli-sperm cell contact. These findings provide a background for similar studies in patients with non-obstructive azoospermia. If reproducible in the latter group, transmeiotic in-vitro differentiation of primary spermatocytes may be useful in cases of complete maturation arrest, whereas the development of culture-specific forms may help select viable spermatids in cases of complete spermiogenesis failure.