Artificial Reproductive Techniques Research Articles

Oocyte collection and outcome following oncologic treatment: a retrospective multicentre study

PurposeThis study assesses fertility treatment outcomes in female patients who had undergone successful oocyte retrieval following cancer therapy.MethodsBetween January 2020 and December 2022, we collected fertility treatment data from six participating centres in Spain and Germany. All patients associated with this data had undergone successful oocyte retrieval following cancer treatment.ResultsWomen had most frequently been diagnosed with a haematological (41.9%), breast (22.6%) or gynaecological malignancy (12.9%); two thirds (67.7%) had previously received a chemotherapy, half a radiotherapy (53.3%) and 45.2% had undergone surgery. On average, 7 years (range 0–28) had passed between cancer treatment and first ovarian stimulation cycle. Forty-nine ovarian stimulation cycles had been conducted on these 31 women between 2004 and 2021 (mean age at first oocyte collection following treatment: 34.8 ± 5.7 years). On average, 7 oocytes were collected per cycle (range 0–26) and 11 were collected per patient (range 0–51). Out of the 190 oocytes collected for immediate use of artificial reproductive technique, 139 were fertilised at a rate of 73%. Live birth rate per fresh transfer was 45% (9/20); no births were reported following cryotransfer (0/10). Mean values of anti-Mullerian hormone (AMH) before stimulation declined with time since treatment; however, oocytes were successfully collected from four women with an AMH of <0.5 ng/ml, although no pregnancies were reported. Ten pregnancies were documented; 3 ended in miscarriage. Two twin and 5 single pregnancies resulted in nine live births. On average, children were carried to term.ConclusionIn this small cohort, oocytes were successfully collected after chemotherapy and radiotherapy, despite—in individual cases—low AMH values. Further studies are needed to enrich the database and ultimately provide appropriate counselling to female cancer patients regarding expectations and ART outcome following cancer therapy.

Effects of flowing water stimulation on hormone regulation during the maturation process of Conger myriaster ovaries.

Conger eel (Conger myriaster) is an economically important species in China. Due to the complex life history of the conger eel, achieving artificial reproduction has remained elusive. This study aimed to explore the effect of water stimulation on hormonal regulation during the artificial reproduction of conger eel. The experiment was divided into four groups: A1 (no hormone injection, still water), A2 (no hormone injection, flowing water), B1 (hormone injection, still water), and B2 (hormone injection, flowing water). The flowing water group maintained a flow velocity of 0.4 ± 0.05m/s for 12h daily throughout the 60-day period. Steroid hormone levels in the serum and ovaries of conger eels were analyzed using UPLC-MS/MS and ELISA on the 30th and 60th days of the experiment. The relative expression levels of follicle-stimulating hormone (FSHβ) and luteinizing hormone (LHβ) in the pituitary were determined by quantitative PCR. The results showed a significantly lower gonadosomatic index (GSI) in B2 compared to B1 (p < 0.05) on the 30th day. FSH was found to act only in the early stages of ovarian development, with water stimulation significantly enhancing FSH synthesis (p < 0.05), while FSHβ gene was not expressed after hormone injection. Conversely, LH was highly expressed in late ovarian development, with flowing water stimulation significantly promoting LH synthesis (p < 0.05). Serum cortisol (COR) levels were significantly higher in the flowing water group than in the still water group (p < 0.05). Furthermore, estradiol (E2) content of B2 was significantly lower than that of B1 on the 30th and 60th day. Overall, flowing water stimulation enhanced the synthesis of FSH in early ovarian development and LH in late ovarian development, while reducing E2 accumulation in the ovaries. In this study, the effect of flowing water stimulation on hormone regulation during the artificial reproduction of conger eel was initially investigated to provide a theoretical basis for optimizing artificial reproduction techniques.

Parental positive affect and negative affect in same- and different-sex parent families: no associations with parental gender and caregiving role.

Positive and negative parental affect influence developing parent-child attachment relationships, especially during infancy as well as children's social-emotional, academic, and behavioral functioning later in life. Increasingly, because both mothers and fathers can play central caregiving roles, the parenting qualities of both parents demand consideration. Therefore, this study investigated whether parental gender and caregiving role were associated with mothers' and fathers' positive affect and negative affect during interactions with their 4-month-old firstborn infant, while determining whether parenting stress, infant temperament, having a singleton/twin, and living in the Netherlands, France, or the United Kingdom were related to parental positive affect and negative affect. In all, 135 different-sex, same-sex male, and same-sex female couples (113 fathers and 157 mothers, comprising 147 primary, and 123 secondary caregivers) who conceived through artificial reproductive techniques were studied. The couples were videorecorded at home while in feeding, cleaning, and playing contexts to assess the levels of positive and negative parental affect. In addition, the couples completed questionnaires about their caregiving role, parenting stress, and the infants' temperament. Mixed linear models indicated that the levels of positive and negative parental affect toward the infant in all contexts were not related to parental gender, caregiving role, the interaction between parental gender and caregiving role, parenting stress, infant temperament, or singleton/twin status. However, the target parental behaviors were related to the country of origin, suggesting differences among Dutch, French, and British parents. Overall, we found no evidence that gender or caregiving roles were associated with the levels of positive and negative affect shown by the parents.

Cryopreservation of the collector urchin embryo, Tripneustes gratilla

The collector urchin, Tripneustes gratilla, is an ecologically important member of the grazing community of Hawai'i's coral reefs. Beyond its ability to maintain balance between native seaweeds and corals, T. gratilla has also been used as a food source and a biocontrol agent against alien invasive algae species. Due to overexploitation, habitat degradation, and other stressors, their populations face local extirpation. However, artificial reproductive techniques, such as cryopreservation, could provide more consistent seedstock throughout the year to supplement aquaculture efforts. Although the sperm and larvae of temperate urchins have been successfully cryopreserved, tropical urchins living on coral reefs have not. Here, we investigated the urchin embryos' tolerance to various cryoprotectants and cooling rates to develop a cryopreservation protocol for T. gratilla. We found that using 1 M Me2SO with a cooling rate of 9.7 °C/min on gastrula stage embryos produced the best results with survival rates of up to 85.5% and up to 50.8% maturation to the 4-arm echinopluteus stage, assessed three days after thawing. Continued research could see cryopreservation added to the repertoire of artificial reproductive techniques for T. gratilla, thereby assisting in the preservation of this ecologically important urchin, all while augmenting aquaculture efforts that contribute to coral reef restoration.

Efficient and repeatable in vitro fertilization in rabbits

Rabbits constitute an interesting model to understand gamete interaction and test novel Artificial Reproductive Techniques, but in vitro fertilization (IVF) is particularly problematic in this species. We have conducted a series of experiments to develop a consistent IVF technique. Initially, we checked viability, acrosome integrity, capacitation and motility in ejaculated sperm purified by a density gradient and incubated at different times in three different media: Tyrode's Albumin Lactate Pyruvate (TALP), human tubal fluid (HTF), and Brackett and Oliphant (BO). Total and progressive motility at 10–24 h and linearity from 3 h onwards was significantly higher in BO medium compared to TALP and HTF. Subsequently, cumulus-oocyte complexes (COCs) collected 10 h after induction of ovulation were incubated with sperm in TALP, HTF or BO for 18 h with or without performing sperm pre-incubation for 6 h. Pronuclear formation rate at 18 h was significantly higher in BO compared to other media (∼84 % vs. 17–22 %) and was not improved by pre-incubation. As COCs recovery rate was low at 10 h after induction of ovulation, COCs were collected at 12 h and co-incubated with sperm in BO. Pronuclear formation rate was similar than those obtained in COCs collected at 10 h (∼85 %), and when embryos were allowed to develop in vitro, the protocol yielded high cleavage and blastocyst rates (91 and 59 %, respectively). In conclusion, ejaculated rabbit sperm purified in a density gradient fertilize efficiently COCs collected at 12 h in BO medium.

The Current Trend of Fertility Preservation in Patients with Cervical Cancer.

Although the incidence of most cancers increases with age, a considerable number of patients receive a diagnosis of cancer during their reproductive years. Young women wishing to get pregnant after cancer treatment should be provided consultation for fertility preservation and possible options. In patients with cervical cancer, hysterectomy is often inevitable because the uterus is located too close to the cervix. For young patients with cervical cancer who desire to get pregnant and whose lesion is confined to the cervix, sparing the uterus and, partially, the cervix should be prioritized as much as possible, while simultaneously ensuring favorable oncologic outcomes. In this review, we explore how to choose an adequate fertility-preserving procedure to achieve a balance between favorable oncologic outcomes and fertility and management during pregnancy after a radical trachelectomy in women with early-stage cervical cancer. For patients who require hysterectomy or radiation, evaluation of the ovarian condition and laparoscopic ovarian transposition followed by the use of artificial reproduction techniques and pregnancy by surrogacy should be discussed as options to achieve a successful pregnancy.

The protective effect of endogenous melatonin on gilthead seabream sperm during cryopreservation

Cryopreservation is widely used for artificial reproduction techniques (ART) but requires good gamete quality to succeed. Considering the endogenously produced melatonin by the fish and its protective role in the organism, the objective of this experiment was to search for the best moment of the day to collect gilthead seabream (Sparus aurata) sperm with better quality and, at the same time, to test the potential protective effect of the exogenous melatonin during sperm freezing and thawing processes. Fish were first sampled every 6 h, corresponding to the beginning of the light period (6 h), mid-light (12h), beginning of the dark period (18 h), and mid-dark (24 h) to determine melatonin concentration in blood plasma by radioimmunoassay (RIA). The analysis showed higher values at 24 h (244 pg/mL) compared with 12 h (99 pg/mL), which allowed the selection of those moments for the next experiment. After, fish were sampled for sperm collection at mid-light (ML, 12 h) and mid-dark (MD, 24 h), and fresh sperm was used to assess gamete quality and for the cryopreservation assay. Sperm samples were cryopreserved to test supplementation of different melatonin concentrations (0.001 mM, 0.01 mM and 0.1 mM) together with a control group without added melatonin. Gamete quality was assessed through spermatozoa concentration and motility (CASA system), cell viability (PI/SYBR-green) and DNA fragmentation (Comet assay). Despite cell viability that was higher at ML, most of the fresh sperm motility parameters did not differ between ML and MD, only linearity (LIN) was enhanced at MD. Nevertheless, in cryopreserved samples, total motility (TM) was significantly higher at MD in all melatonin treatments, control, and fresh samples, revealing an endogenous night-effect. Moreover, spermatozoa concentration was also higher at MD (28.9 × 109/mL) than at ML (20.7 × 109/mL). Supplemented melatonin did not confer extra protection to gilthead seabream sperm during cryopreservation since the tested concentrations did not differ between the control in any sperm quality test. It is here suggested that endogenously produced melatonin may contribute to the improvement of some gamete quality parameters at mid-dark, allowing the aquaculture sector to select better sperm quality in a non-invasive way by choosing it as the best moment of the day for sperm collection.

Estimates of the Effective Population Size and Genetic Structure of the Critically Endangered Ship Sturgeon (Acipenser nudiventris) in the Chinese Section of the Ili River

Ship sturgeon (Acipenser nudiventris) is a critically endangered fish that is listed on the International Union for Conservation of Nature’s Red List of Threatened Species. Sixteen individuals from the Chinese section of the Ili River were genotyped using genome re-sequencing technology. By applying a genomic relatedness estimation with 1,527,694 genome-wide SNP markers, we found that the coancestry coefficients showed a high level of relatedness between individuals. The effective population sizes over 500 generations were estimated, and this showed that the effective population sizes began to dramatically decline from about 14,840 to 171 individuals when going back four generations from the current population. Artificial reproduction techniques guided by genomic relatedness may be a valuable approach to the conservation of this critically endangered fish species.

P-096 Prediction of fertilization success by voltage-dependent intracellular Ca2+ dynamics in non-normozoospermic human semen samples undergoing in vitro fertilization: preliminary results

Abstract Study question Does the change in intracellular calcium levels evoked by artificial membrane depolarizations predict fertilization success in a non-normozoospermic semen sample undergoing assisted reproductive techniques? Summary answer The increase in the intracellular calcium after artificial plasma membrane depolarization correlates negatively with fertilization rates in conventional IVF but not with ICSI What is known already Sperm capacitation-related features, like the regulation of the intracellular calcium concentration ([Ca2+]i) and the hyperpolarization of the plasma membrane potential (Vm), have been proposed as predictors of success during artificial reproduction techniques (ART). Intriguingly, upon Vm depolarization, sperm exhibit a rapid and transitory increase in the [Ca2+]i controlled, likely, by the activation of the sperm-specific calcium channel CatSper. The regulation of CatSper activity is essential for sperm function and is crucial for fertilization. Therefore we wondered if the Vm depolarization-dependent rise in the [Ca2+]i can be used to predict fertilization success rates in non-normozoospermic semen samples of men undergoing ART. Study design, size, duration This prospective study employed unidentified semen samples from 29 non-normozoospermic men (37.8 ± 5.6 years old) undergoing fertility treatment from March 2022 to the present at CITMER Reproductive Medicine, Mexico City. Frozen semen samples and procedures with less than 3 oocytes were excluded. Ten normozoospermic semen samples were analyzed as a control. The depolarization-dependent [Ca2+]i changes were evaluated by flow cytometry under four different Vm conditions: resting (variable among samples), –64, –46, and –32 mV. Participants/materials, setting, methods Semen samples were obtained on the same day as oocyte retrieval. Sperm cells were washed and incubated in capacitating HTF media for further use in conventional IVF or ICSI procedures. When available, the surplus cells were stained with vitality, Ca2+, and Vm-sensitive fluorescent dyes. Fluorescence changes were evaluated by AccuriC6 flow cytometer. The kinetical analysis of the transitory [Ca2+]i increase as well as the statistical comparisons were performed using custom-made R programming language code. Main results and the role of chance Analysis of resting Vm showed that normozoospermic semen samples presented more depolarized Vm than non-normozoospermic controls (–75.7 ± 6.9 vs –67.3 ± 10.5 mV, respectively, p = 0.02). The former group of semen samples was either used for conventional IVF (n = 11) or ICSI (n = 14). The semen samples in these last categories showed a tendency to present a more depolarized Vm than non-normozoospermic samples (–69.9 ± 8.8 and –66.6 ± 11.3 mV, respectively). No differences were seen in the resting Ca2+ levels. We then induced a Vm hyperpolarization by adding the K+ ionophore, valinomycin followed by increasing concentrations of extracellular K+. Each depolarizing stimulus evoked a fast and transitory increase in the [Ca2+]i. We then calculated seven different kinetic parameters of such responses: basal Ca2+ level, increase and decrease velocity, maximum Ca2+ reached, response durability, and area under the curve (AUC). The basal Ca2+ level reached after depolarizing stimuli correlated negatively (R = –0.64, p = 0.03) with the conventional IVF fertilization ratio (number of zygotes/total number of mature oocytes) when the Vm was clamped either to –64 and –46 mV. A negative and significant correlation (R = –0.76, p = 0.006) was seen for the AUC analysis at –64 mV. No relevant correlations were found in the ICSI samples. Limitations, reasons for caution This is an in vitro study, and caution must be taken when extrapolating these results in vivo. The preliminary nature of the results implies a necessity to increase the number of analyzed semen samples (in progress). Wider implications of the findings Further research into the molecular regulation of the CatSper channel could offer promising alternatives for clinicians to appropriately choose between IVF and ICSI for each couple. This protocol is fast and simple to perform in a laboratory equipped with a flow cytometer or any fluorescence-based reader. Trial registration number NA

Spontaneous haemoperitoneum in pregnancy: Nationwide surveillance and Delphi audit system.

To evaluate the incidence, diagnostic management strategies and clinical outcomes of women with spontaneous haemoperitoneum in pregnancy (SHiP) and reassess the definition of SHiP. A population-based cohort study using the Netherlands Obstetric Surveillance System (NethOSS). Nationwide, the Netherlands. All pregnant women between April 2016 and April 2018. This is a case study of SHiP using the monthly registry reports of NethOSS. Complete anonymised case files were obtained. A newly introduced online Delphi audit system (DAS) was used to evaluate each case, to make recommendations on improving the management of SHiP and to propose a new definition of SHiP. Incidence and outcomes, lessons learned about clinical management and the critical appraisal of the current definition of SHiP. In total, 24 cases were reported. After a Delphi procedure, 14 cases were classified as SHiP. The nationwide incidence was 4.9 per 100 000 births. Endometriosis and conceiving after artificial reproductive techniques were identified as risk factors. No maternal and three perinatal deaths occurred. Based on the DAS, adequate imaging of free intra-abdominal fluid, and identifying and treating women with signs of hypovolemic shock could improve the early detection and management of SHiP. A revised definition of SHiP was proposed, excluding the need for surgical or radiological intervention. SHiP is a rare and easily misdiagnosed condition that is associated with high perinatal mortality. To improve care, better awareness among healthcare workers is needed. The DAS is a sufficient tool to audit maternal morbidity and mortality.

Metabolic Mechanisms and Potential Therapeutic Targets for Prevention of Ovarian Aging: Data from Up-to-Date Experimental Studies.

Female infertility and reproduction is an ongoing and rising healthcare issue, resulting in delaying the decision to start a family. Therefore, in this review, we examine potential novel metabolic mechanisms involved in ovarian aging according to recent data and how these mechanisms may be addressed through new potential medical treatments. We examine novel medical treatments currently available based mostly on experimental stem cell procedures as well as caloric restriction (CR), hyperbaric oxygen treatment and mitochondrial transfer. Understanding the connection between metabolic and reproductive pathways has the potential to offer a significant scientific breakthrough in preventing ovarian aging and prolonging female fertility. Overall, the field of ovarian aging is an emerging field that may expand the female fertility window and perhaps even reduce the need for artificial reproductive techniques.

Understanding Progestins: From Basics to Clinical Applicability.

Progestin is a term used to describe a synthetic progestogen. The activity and potency of synthetic progestins are mostly evaluated via parameters associated with their endometrial effects, which are related to their interactions with progesterone, estrogen, androgen, glucocorticoid, and mineralocorticoid receptors. The chemical structure of progestins is the key to understanding their interactions with these receptors and predicting the other effects associated with these drugs. Due to their endometrial effect, progestins are used for different gynecological conditions, such as endometriosis, contraception, hormonal replacement therapy, and artificial reproduction techniques. This review is focused on improving our knowledge of progestins (from their history and biochemical effects related to their chemical structures to clinical applications in gynecological conditions) in order to improve clinical practice.

Effectiveness of Parental Preparedness Package among Couples Conceived with Artificial Reproductive Techniques.

The problem of infertility is experienced as a crisis or major life stressor with potentially serious negative effects on the couple's relationship and preparation toward the parenthood. According to Sharlip et al., it has been estimated that about 15% of couple would have trouble in conception, and at present, 48.5 million couples experience infertility globally. To decrease the stress among parents to be and to promote healthy parenting for bringing out healthy parent-child relationships, an intervention of Parent Education Program was planned and executed. A study was conducted with the aim to assess the effectiveness of parental preparedness package (PPP) on physical health among couple conceived with artificial reproductive techniques (ART). The study was conducted to evaluate the effectiveness of PPP in fifty couples conceived with ART at Dayanand Medical College and Hospital, Ludhiana. Quantitative approach was used to collect the data of couples by doing pre- and postinterventional assessment of physical health of couple using the Structured Physical Health Assessment Scale. Analysis was done with the help of descriptive and inferential statistics. The study revealed that mean score of physical health of couple (both husband and wife) increased significantly after the intervention which infers the effectiveness of PPP (P = 0.001). Our study concludes that PPP should be incorporated in the antenatal care protocol in all couples who conceive using ART.

Surrogacy (Regulation) bill 2020 and its implications on the reproductive tourism industry in India

Surrogacy in India is an unfettered and unregulated practice which is being carried out blatantly without any legal sanction. The guidelines issued by the Indian Council of Medical Research to regulate clinics running Surrogacy arrangements under the umbrella of Artificial Reproductive Technique are the current driving force of this Fertility Tourism Industry. The codified law is yet to be adopted and implemented. The growth in the assisted reproductive technology (ART) methods is the recognition of the fact that infertility as a medical condition is a huge impediment in the overall well-being of couples and cannot be overlooked, especially in a patriarchal society like India. A woman is respected as a wife only if she is a mother of a child so that her husband's masculinity and sexual potency is proved and the lineage continues. The practice of nourishing another's fetus in the womb is known as surrogacy. Surrogacy has been the most contentious use of ARTs. These include the possibility of finding substitutes not only for parental genetic material but also for the womb. Surrogacy is a tripartite agreement which includes the intending parents, the surrogacy clinics, and the surrogate woman who agree to commission a pregnancy on behalf of another woman, ready to relinquish all her rights on the child born out of the arrangement for a monetary remuneration. The practice of surrogacy involves social, moral, legal, ethical, and scientific issues in an attempt to harmonize conflicting interests. This paper will focus on the new Surrogacy Bill 2020 and its implications on the medical infertility tourism industry in India.

A Rare Case of Shared Delusion of Pregnancy in a Couple Undergoing Fertility Treatment

Pseudocyesis or phantom pregnancy is a false belief of being pregnant that is associated with objective signs and reported symptoms of pregnancy, which may include abdominal enlargement, reduced menstrual flow, amenorrhea, subjective sensation of fetal movement, nausea, breast engorgement and secretions, and labor. Though it is a very rare condition, it is often seen in women undergoing fertility treatment in developing countries. Isolated pseudocyesis in women and sometimes men have been reported before, but there are very few reports of shared delusions of pregnancy by a couple. No such report has ever been made before from Nepal. Therefore, this case report seeks to associate psychiatric/psychological, gynecological, and neuroendocrine mechanisms leading to the emergence of shared delusion of pregnancy (folie à deux) in a Nepali couple undergoing artificial reproductive techniques for the management of infertility. In this case, the couple firmly believed in their pregnancy against all medical evidence.

Effect of ovarian growth factors on ultra-structural maturation in frozen human immature oocytes after in vitro maturation: a comparative study

BackgroundIn artificial reproductive technique (ART), nearly 20% of human oocytes are immature in the germinal vesicle (GV) phase. Consequently, the best method for reserving them is cryopreserving GV oocytes, and in vitro maturation (IVM) is recommended. The aim of this study was to determine the ultrastructure characteristics of fresh and vitrified immature human oocytes after in vitro maturation in conditioned mediums.MethodsThis study was a comparative laboratory study carried out in 2018 at Afzalipur Infertility Center in Kerman. 170 fresh and 198 vitrified GV oocytes were cultured within three IVM mediums; α-mem as control medium, α-mem supplemented with human bone marrow mesenchymal stem cells (BM-MSCs) and α-mem supplemented with ovarian growth factors (O.F). After 48 h, the maturation rate and morphological feature of IVM oocytes [132 fresh IVM (fIVM) and 134 vitrified IVM (vIVM)] were evaluated. For the ultrastructure study, 10 IVM oocytes from each medium were compared with 10 fresh in vivo oocytes cancelled from ART.ResultsThe survival rate of vitrified GV oocyte after thawing was 88.88%. The oocyte maturation rate was reduced in vIVM compared to the fIVM group (76.33% vs. 77.95%); the highest oocyte maturation rate in the O.F fIVM and lowest in α-mem vIVM (82.35% vs. 71.42%). The lowest number of cortical granules was observed in α-mem vIVM, but the greatest presence of M-SER aggregates was in O.F fIVM. In vIVM oocytes, the oolemma contained irregular little microvillus organization.ConclusionsThe O.F mediums have shown the highest maturation which defends the oocyte ultra-structural conservation.

A Case Report on the Impact of a Lean Poly Cystic Ovarian Syndrome on Fertility and Potential Benefit of Ayurveda Regime

Despite of advances in Artificial Reproductive Techniques (ART), female infertility is the predominant health issue faced by approximate 15% couples globally.Out of the ovarian factors responsible for infertility, PCOS is a burning issue worldwide affecting female fertility and creating endocrine level disturbances. Case presentation: We are reporting a case of primary infertility presenting with Lean phenotypical features and USG findings of PCOS. She underwent conventional Hormonal therapy for two years and single cycle of IVF which resulted in failure. On evaluating the parameters in view of Ayurveda, a diagnosis of Anapatya Vandhya with Artavakshaya and Karshya was made. Intervention: Therapeutic strategy included Shamana therapy for three months followed by Shodhana Therapy involving Uttarabasti (Intrauterine instillation of drug), Ksheerabasti and Nasya Karma. Outcome: Following treatment, 4 kg gain in body weight, moderate relief in acne and substantial improvement in duration and flow of menstruation was observed and the patient conceived in the 4th month of treatment course. Conclusion: This case report presents a successful outcome of Ayurveda regime planned on the basis of Ayurveda diagnosis in a rare case of Primary infertility associated with Lean PCOS

Preconception Counseling in Patients with Hypothyroidism and/or Thyroid Autoimmunity.

Preconception counseling is an essential tool for preventing adverse pregnancy outcomes associated with thyroid dysfunction. The high prevalence of thyroid disease among women of reproductive age, and the increased risk of adverse pregnancy outcomes associated with thyroid dysfunction, emphasize the necessity for well-established screening and treatment criteria in the preconception period. We therefore conducted a literature review for relevant information on the screening, diagnosis and treatment of subclinical and overt hypothyroidism in women seeking pregnancy. While screening for thyroid disease is recommended only in the presence of risk factors, iodine supplementation should be recommended in most regions, with higher doses in areas with severe deficiency. Known hypothyroid women should be counseled about increasing their levothyroxine dose by 20–30% in the case of suspected or confirmed pregnancy (missed menstrual cycle or positive pregnancy test). Treating subclinical hypothyroidism appears to be beneficial, especially in the presence of autoimmunity or in patients undergoing artificial reproductive techniques. Regarding the management of TPOAb negative SCH women or euthyroid women with positive TPOAb, further research is necessary in order to make evidence-based recommendations.

P-257 Nanosphere-based assessment of zona pellucida permeability in humans and mammalian models

Abstract Study question What is the maximum particle size that can traverse through human zona pellucida (ZP) and that of other mammals? Summary answer Human oocytes ZP is impermeable to 40-100 nm nanospheres. ZP of other mammals is impermeable to 100 nm particles, and permeability changes along preimplantation development. What is known already ZP is a porous structure that protects oocytes and early embryos, being permeable to nourishing and signaling substances while blocking the entrance of immune cells and viruses into the perivitelline space. Size-dependent ZP permeability is poorly known and may differ between species, as in humans and rabbits ZP it is composed by 4 proteins (ZP1-4), while bovine and murine ZPs contain only 3 proteins (ZP2-4 in bovine, ZP1-3 in mice). Understanding permeability is crucial to determine embryo susceptibility to viral infections or large biological particles such as exosomes, and to assess possible damages inferred by artificial reproductive techniques. Study design, size, duration The objective has been to determine ZP permeability in mice, bovine, rabbit (wild-type or lacking ZP4) or human oocytes and/or embryos based on particle size. The assay was performed in 3 replicates of 3 samples per stage: mouse samples (oocytes, zygotes, 2-cell embryos and blastocysts) collected in vivo, bovine samples (oocytes, zygotes, 4-8 cell embryos and blastocysts) produced in vitro, rabbit samples (zygotes) collected in vivo and immature human oocytes discarded for clinical use. Participants/materials, setting, methods Permeability test consisted of incubating oocytes and embryos in 0.005 % solutions of carboxylate-modified polystyrene nanospheres (20, 40 and 100 nm) loaded with a fluorophore emitting at a 505/515 nm for 30 min. Following incubation and washing, samples were observed under a structured illumination fluorescence microscope to determine if particles are present inside ZP (i.e., ZP is permeable to that size of particle) or fluorescence cannot be detected (i.e., particles unable to traverse ZP). Main results and the role of chance ZPs of all species and stages blocked efficiently the pass of 100 nm-sized nanospheres. 40 mm nanospheres were blocked by the ZP of oocytes and zygotes in all species with the exception of rabbit zygotes lacking ZP4. ZP permeability changed during early development, as ZP from murine and bovine cleaved embryos and blastocyst were permeable to 40 nm-sized particles. Finally, with the exception or WT rabbit zygotes, ZP from all oocytes and embryos analyzed were permeable to 20 nm nanospheres. As a fluorescence control, murine and bovine oocytes without ZP were exposed to nanospheres of all sizes, which were clearly visualized attached to the oolema or into the ooplasm. Limitations, reasons for caution Experiments on cleavage and blastocysts stages were performed on animal models, so whether human ZP permeability changes following fertilization remains unknown. Wider implications of the findings Mammalian ZP acts as a selective barrier during early embryo development. Understanding particle size-dependent permeability is crucial to determine embryo susceptibility to viral infection or to signaling mediated by large particles such as extracellular vesicles and exosomes and to assess the impact of oocyte manipulation techniques. Trial registration number Not applicable

Comparison of Efficacy Between Vaginal Sildenafil and Granulocyte-Colony Stimulating Factor (G-CSF) in Improving Endometrial Thickness (ET) in Infertile Women.

BackgroundInfertility is presently an emanating preventive medicine issue with some severe societal repercussions associated with it. In India, approximately a score percent of couples bear the burden of infertility. Moreover, the declining fertility rates despite effective artificial reproductive techniques and increasing development of modern reproductive medicine from the last two censuses pose an alarm to the demographic progression data. Many studies have highlighted the importance of shifting the research focus to endometrial receptivity for increasing clinical pregnancy. Objective This research aims to compare the efficacy of treatments of vaginal sildenafil citrate and granulocyte-colony stimulating factor (G-CSF) intrauterine injection in increasing endometrial thickness (ET).Methodology This was a randomized control trial (RCT) conducted over a two months period. Women seeking infertility treatment were recruited from the hospital's gynecological outpatient department (OPD). After the subjects gave informed consent, their history, clinical examination, and investigations were assessed. From the sixth day of the menstrual cycle, group A and group B had serial trans-vaginal ultra-sonographic evaluations for baseline endometrial thickness measurements. From day six to day 12 of the menstrual cycle, patients of group A were requested to self-administer per vaginal sildenafil citrate 25 mg every six hours. ET was evaluated sonographically on day 12 and day 14 of their menstrual cycle. Patients of group B received G-CSF 300 mcg/ml as intrauterine instillation on day 10 and were evaluated sonographically on day 12 and day 14 of their menstrual cycle. Patients then underwent additional therapy in the form of intrauterine injection (IUI), intracytoplasmic sperm injection with/without embryo transfer (ICSI/ET), or a natural cycle. Paired as well as unpaired t-tests were applied to the study groups to detect significant differences in the measurement of endometrial thickness before and after treatment.ResultsIt was noticed that both sildenafil and G-CSF are agents for increasing endometrial thickness. The mean increase in endometrial thickness in the sildenafil treated group was 3.87 mm, while the mean increase in endometrial thickness in G-CSF treated group was 3.27 mm.Conclusion This study has evidence of better results in improving endometrial thickness in infertile women by using vaginal sildenafil with respect to endometrial growth with an intrauterine infusion of granulocyte colony-stimulating factor (filgrastim, G-CSF).