Sequential (two-step) day 3/day 5 frozen-thawed embryo transfer: does it improve the pregnancy rate of patients suffering recurrent implantation failure?

The best time of endometrial receptivity is the missing part of the implantation puzzle in patients with recurrent in vitro fertilization (IVF) failure. There are various treatment plans and strategies to meet the best endometrial timing for implantation. However, the lack of synchronization of the good-quality embryo with the patient's individual "window of implantation" is the hypothesis for most IVF failures so far. Sequential embryo transfer (ET) theoretically extends the availability time of embryos on the window of implantation. The study aimed to evaluate the improvement of pregnancy rate in sequential (two-step) frozen-thawed embryo transfer (FET) on day 3/day 5 in individuals who suffer from repeated IVF failures. This randomized controlled trial study was done in a university-affiliated infertility center for women with repeated consecutive IVF failures. Two hundred women aged 20-39 years who met our inclusion criteria were included in the study between January 2020 and September 2021. Participants were allocated with a 1:1 ratio to either sequential (two-step) ET on day 3/day 5 (study group, n=100) and conventional day 5 FET (n=100, control group). The frozen-thawed embryos were transferred to hormone replacement therapy-prepared endometrium in both groups. The primary outcomes were clinical pregnancy and implantation rates. The secondary outcomes were early pregnancy loss and multiple pregnancies. The demographic and clinical characteristics of the two groups were comparable. Clinical pregnancy rates were significantly higher in the sequential (two-step) FET group (40%) compared to the day 5 group (19%) (P<0.001). The sequential transfer of frozen-thawed embryos on day 3/day 5 was more effective than regular day 5 for patients suffering from repeated IVF failure.

Effective dosage of growth differentiation factor-9ß in folliculogenesis and angiogenesis in the sheep ovarian tissues grafted onto chick embryo chorioallantoic membrane.

AIM: Scientists have tried to culture and transplant the ovarian tissues (OT), to preserve fertility in cancer patients. However, one of the main limitations to the applicability of this technique is the folliculogenesis disruption after transplantation. Due to the roles exerted by growth differentiation factor-9ß (GDF9ß), we decided to determine the most effective dose of GDF9ß on promotion of folliculogenesis and angiogenesis in sheep OT grafted onto the chick chorioallantoic membrane (CAM). METHODS: Fresh sheep OT were grafted onto the CAM for 5 days, and divided into four groups based on the addition of increasing doses of GDF9ß (0, 150, 200 and 250 ng/mL). Following culture, histological (hematoxylin and eosin [H&E] staining) and immunohistological studies (Ki-67) were done. Fibrotic and necrotic regions were measured using MICROVISIBLE software. For comparing the follicle development rates between the groups as well as differences in the Ki-67-positive follicles, analysis of variance was applied. RESULTS: In both 200 and 250 ng/mL GDF9ß groups, significantly higher rates of intermediary and primary follicles were observed, also the numbers of good quality follicles increased in the aforementioned groups and the rates of fibrotic and necrotic areas decreased. Moreover, in the 200 and 250 ng/mL GDF9ß groups, the number of capillaries and the proliferative activity increased. The lower dose of GDF9ß (150 ng/mL) neither activated the primordial follicles nor lead to an increase in the number of growing follicles. CONCLUSION: Addition of high dosages of GDF9ß to the OT, grafted onto the CAM resulted in higher folliculogenesis and better transplantation features due to improvement in angiogenesis.

Cryopreservation of Low Number of Human Spermatozoa; Which is Better: Vapor Phase or Direct Submerging in Liquid Nitrogen?

Cryopreservation and subsequent survival of semen samples with low numbers of human spermatozoa in assisted reproductive technology (ART) facilities can be challenging. The aim of this study was to compare the quality of warmed human spermatozoa following vitrification using direct submerging (DS) in liquid nitrogen (LN2) or with LN2 vapour (V). Normozoospermic ejaculates were prepared by the swim-up technique and the motile sperm fraction was divided into three groups: (i) fresh (control); (ii) DS methods in LN2 (DS Group); or (iii) cryopreserved in V (Group V). Cryopreservation was performed in a small volume using a Cryotech device. After warming, sperm parameters (motility, viability, acrosome, DNA fragmentation and chromatin integrity) were assessed using cytochemical assays. Progressive motility, viability, chromatin integrity and DNA fragmentation differed significantly after warming when compared with the control. Sperm progressive motility and total motility rates were significantly higher in the DS Group compared to Group V. However, normal morphology, acrosome integrity and DNA damage were not significantly different between two groups. Also, sperm chromatin condensation using chromomycin-A3 (CMA3) and Aniline Blue (AB) staining showed fewer alterations in the DS Group compared to Group V. The rates of spermatozoa with an intact acrosome decreased significantly after thawing from 81.30 ± 6.76% to 68.84 ± 14.70% in the DS Group and to 60.92 ± 8.06% in Group V. Cryopreservation of a small number of spermatozoa with the DS method showed superior outcomes with regard to motility, viability and chromatin configuration.

GDF9-ß promotes folliculogenesis in sheep ovarian transplantation onto the chick embryo chorioallantoic membrane (CAM) in cryopreservation programs.

PURPOSE: Ovarian tissue (OT) cryopreservation is a treatment option for fertility preservation among young cancer patients. However, the procedure may involve a reduction in the GDF9-ß expression and a delay in follicular growth after thawing and transplantation. The aim of this study was to evaluate whether supplementation of GDF9-ß can compensate the reduction of this factor during the cryopresevation process and promote folliculogenesis after transplantation of thawed sheep ovarian tissue. METHODS: Sheep OT was cryopreserved using two methods of vitrification and slow freezing. Fresh and thawed OTs were then transplanted onto chick embryo chorioallantoic membrane (CAM) and then divided into two groups based on the addition of GDF9-ß to the grafted tissue. After 5 days of culture, both histological and immunohistological (Ki-67) assessments were performed to evaluate follicular structure, development, and proliferation. The fibrotic and necrotic areas were measured using MICROVISIBLE software. RESULTS: Folliculogenesis took place in all culture groups, but was significantly improved only in the +GDF9-ß cultured group. Also, better follicular structure was preserved in the aforementioned group (p < 0.05). When GDF9-ß was supplemented to the culture medium, more neovascularization (p < 0.05) and better transplantation (p > 0.05) was observed. Furthermore, the areas of fibrosis and necrosis were lower in this group rather than the controls. Follicular proliferative activity was significantly higher only in the slow freezing +GDF9-ß cultured group. CONCLUSIONS: GDF9-ß, as a stimulatory factor, not only promoted the folliculogenesis in the fresh ovarian transplant, but also compensated for its reduction during the cryopreservation process.

Evaluation of sheep ovarian tissue cryopreservation with slow freezing or vitrification after chick embryo chorioallantoic membrane transplantation.

The aim of our investigations was to compare the effectiveness of two methods for cryopreservation of sheep ovarian tissue, slow freezing and vitrification. The quality of cryopreserved tissues was evaluated after 5 days of thawing and chorioallantoic membrane (CAM) transplantation. Follicular structure, stromal integrity and neovascularization were assessed. The areas of fibrosis and necrosis were measured using MICROVISIBLE software, and proliferation was assessed with Ki-67 immunostaning. After 5 days of culture, the proportion of primordial follicles decreased, whereas the primary and intermediary follicles increased insignificantly (p > .05). Only necrosis in the vitrified culture group increased significantly (p < .05). It was established also that 5 days CAM culture was not suitable methodology for detection of folliculogenesis. Follicular quality decreased after culture, but was better in fresh and slow frozen tissues than after vitrification (p < .05). Cellular proliferative activity fell, but it preserved to some extent in all groups. In conclusion, follicles was preserved better in grafted tissue after slow freezing than vitrification and stroma was more susceptible to ischemia in vitrified rather than conventional freezing in this view. Vitrification may not be a suitable alternative to the slow freezing.

Embryo Cryopreservation Following In-Vitro Maturation for Fertility Preservation in a Woman with Mullerian Adenosarcoma: A Case Report.

In-vitro maturation (IVM) of the immature oocytes recovered from the surgically removed ovarian tissue has been considered as a process for fertility preservation in patients with cancer. Fertility preservation for a woman with Mullerian adenocarcinoma. A 37-year-old woman with Mullerian adenocarcinoma was a candidate for ovarian resection. The immature oocytes were retrieved after ovarian resection from a 37-year-old woman with Mullerian adenocarcinoma. The oocytes underwent IVM and were fertilized using intracytoplasmic sperm injection (ICSI). Two healthy embryos were cryopreserved for future use. The immature oocytes from the ovarian tissue can be matured with IVM for generation of embryos after ICSI. The embryos can be vitrified using routine methods for fertility preservation in young women with cancer.

In-Vitro Application of Pentoxifylline Preserved Ultrastructure of Spermatozoa After Vitrification in Asthenozoospermic Patients.

PURPOSE: To evaluate the effect of in vitro application of pentoxifylline (PX) on sperm parameters and ultrastructure after vitrification in asthenozoospermic patients. MATERIALS AND METHODS: A total of 30 asthenozoospermic semen samples (aged 25-45 years) were divided into four groups before vitrification, after vitrification, control (without PX) and experimental (with PX). In experimental group, each sample was exposed for 30 min to 3.6mmol/l PX and the control group without any treatment apposing in 370C for 30 min. After incubation, the samples were washed and analyzed again. Vitrification was done according to straw method. Eosin-nigrosin and Papanicolaou staining were applied for assessment of sperm viability and morphology, respectively. The samples without PX and post treatment with PX were assessed by transmission electron microscopy (TEM). RESULTS: A significant decrease in sperm motility (P ≤ .001), morphology (11.47 ± 2.9 versus 6.73 ± 2.01) and viability (73.37 ± 6.26 versus 54.67 ± 6.73) was observed post vitrification, but sperm motility (19.85 ± 4.75 versus 32.07 ± 5.58, P ≤ .001) was increased significantly following application of PX. This drug had no significant (P >.05) detrimental neither negative effect on ultrastructure acrosome, plasma membrane and coiled tail statues of spermatozoa. CONCLUSION: Vitrification had detrimental effects on sperm parameters, but PX reversed detrimental effects on sperm motility. However, PX had no alteration on ultrastructure morphology of human spermatozoa after vitrification.

Transplantation of differentiated umbilical cord mesenchymal cells under kidney capsule for control of type I diabetes in rat.

Nowadays, stem cells have been introduced as an appropriate source of regenerative medicine for treatment of type I diabetes. Human umbilical cord matrix-derived mesenchymal cells (hUCMC) have successfully been differentiated into insulin producing cells. The isolated hUCM cells were characterized by the expression of stem cell surface markers and by differentiation into adipocytes and osteocytes. The hUCMCs were cultured with different concentrations of neural conditional medium (NCM) and were induced to differentiate into insulin producing cells (IPCs). As 60% NCM concentration resulted in higher nestin and PDX1 expression, the cells were first exposed to 60% NCM and were then induced for IPCs differentiation. PDX1 and insulin gene expression was evaluated in the treated cells. Also, the secretion capacity of the IPCs was assessed by glucose challenge test. IPCs were transferred under the rat kidney capsule. Blood glucose level, weight gain and immunohistochemistry assessments were done in the treated animals. hUCMC expressed mesenchymal cell surface markers and successfully differentiated into adipocytes and osteocytes. Higher NCM concentration resulted in higher PDX1 and nestin expression. The IPCs expressed insulin and PDX1. IPCs were detectable under the kidney capsule 2 months after injection. IPCs transplantation resulted in a sharp decline of blood sugar level and less weight loss. Differentiated hUCM cells could alleviate the insulin deprivation in the rat model of type I diabetes. In addition, higher NCM concentration leads to more differentiation into IPCs and more nestin and PDX1 expression. Kidney capsule can serve as a suitable nominee for IPCs transplantation.

Results from adding recombinant LH for assisted reproductive technology treatment: A randomized control trial.

BACKGROUND: Based on classical two-cell, two-gonadotropin theory, in the follicle, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) put on their main effects on the granulosa and theca cells. LH is essential for androgens production. Androgens are used for estradiol production by granulosa cells. Profound suppression of LH concentrations in some normogonadotropic patients can cause several adverse effects. OBJECTIVE: The main clinical purpose of this study was that normoresponder women treated with controlled ovarian super ovulation for IVF or ICSI may benefit from co-administration of rLH. MATERIALS AND METHODS: 40 patients who were candidates for assisted reproductive technology (ART) were randomly selected. In all patients long luteal protocol was used for ovulation induction. Patients were randomly divided into two groups: Group 1 (n=20) with standard long protocol (GnRH agonist) and r-FSH alone, Group 2 (n=20) with standard long protocol (GnRH agonist) and r-FSH with r-LH. RESULTS were statistically analyzed and compared in two groups. RESULTS: The number of retrieved oocytes, mature oocytes, cleaved embryos, transferred embryos, estradiol levels in Human chorionic gonadotropin (hCG) administration day, implantation rate and clinical pregnancy rate in group 2 were higher but not significantly different. CONCLUSION: Administration of rLH in late follicular phase had no beneficial effect on outcomes in young women with mean age of 31 years. Maybe a greater sample size should be used to see the effects more accurately; also it is possible that rLH will be useful in older patients. Registration ID in IRCT: IRCT201304302575N4.