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The preservation of fertility in cancer patients is a crucial aspect of modern reproductive medicine. Amenorrhea and infertility often occur after cancer therapy, worsening the quality of life. Cryopreservation of oocytes in young cancer patients is a therapeutic option for preserving fertility. A prospective study was conducted on 508 cancer patients who underwent oocyte cryopreservation to preserve fertility between 1996 and 2021 including the COVID-19 pandemic period. Patients underwent ovarian stimulation, followed by egg retrieval, and oocytes were cryopreserved by slow freezing or vitrification. Sixty-four thawing/warming cycles were performed. Survival, fertilization, pregnancy, and birth rate over the thawing/warming cycles were obtained. The data were compared with those from a group of 1042 nononcological patients who cryopreserved supernumerary oocytes. An average of 8.8 ± 6.9 oocytes were retrieved per cycle, and 6.1 ± 4.2 oocytes were cryopreserved. With their own stored oocytes, 44 patients returned to attempt pregnancy. From a total of 194 thawed/warmed oocytes, 157 survived (80%). In total, 100 embryos were transferred in 57 transfer/cycles, and 18 pregnancies were achieved. The pregnancy rate per transfer and pregnancy rate per patient were 31% and 41%, respectively. No statistically significant differences were observed between oncological patients and nononcological patients. A total of 15 babies were born from oncological patients. Children born showed normal growth and development. One minor malformation was detected.

Background: Sexual abstinence is considered one of the several factors that influence sperm quality. Recent studies show that a shortening of the abstinence period could be beneficial mostly in oligoasthenoteratozoospermic (OAT) patients., Objective: Retrospective study to verify the efficacy of a second semen sample after a short abstinence to treat severe OAT infertile patients., Materials and Methods: 127 couples treated between May 2014 and May 2018 were divided into two groups. Study Group 1 (75 cycles): severe OAT characteristics: count <0.2 × 10 6 /mL no progressive motility; count ≥0.2 × 10 6 /mL and no total or progressive motility; 0% normal morphology; a second semen sample was requested after abstinence of 2 h. Control Group 0 (52 cycles): normozoospermic or mild OAT; only one sample was requested. Intracytoplasmic sperm injection was utilized in all cases., Results: All semen parameters were significantly different between Group 0 vs both samples of Group 1 (p < 0.001), excluding volume between Group 0 and 1st sample of Group 1 (p = 0.682). The comparison between 1st and 2nd samples from Group 1 showed significant differences in volume, total and progressive motility and morphology (p < 0.001, p < 0.001, p < 0.020) but not in total sperm count (p = 0.970). Fertilization, pregnancy rate/transfer, implantation and miscarriage rates were 85.9% and 61.1% (p < 0.001), 30.6% and 35.8% (p = 0.700), 17.5% and 24.0 (p = 0.292), 20.0% and 25.0% (p = 0.017) in Group 0 and Group 1 respectively., Discussion and Conclusion: The results show that a short abstinence in severe OAT patients allows us to obtain spermatozoa with better motility. The request for a second semen sample in couples with extreme semen parameters is a valid and simple strategy that helps to achieve the same probability of pregnancy compared to a Control Group. Furthermore, it allows us to utilize fresh spermatozoa avoiding the need to resort to cryopreserved reserves or testicular surgery., (© 2021 American Society of Andrology and European Academy of Andrology.)

Purpose: The main purpose and research question of the study are to compare the efficacy of high-security closed versus open devices for human oocytes' vitrification., Methods: A prospective randomized study was conducted. A total of 737 patients attending the Infertility and IVF Unit at S.Orsola University Hospital (Italy) between October 2015 and April 2020 were randomly assigned to two groups. A total of 368 patients were assigned to group 1 (High-Security Vitrification™ - HSV) and 369 to group 2 (Cryotop® open system). Oocyte survival, fertilization, cleavage, pregnancy, implantation, and miscarriage rate were compared between the two groups., Results: No statistically significant differences were observed on survival rate (70.3% vs. 73.3%), fertilization rate (70.8% vs. 74.9%), cleavage rate (90.6% vs. 90.3%), pregnancy/transfer ratio (32.0% vs. 31.8%), implantation rate (19.7% vs. 19.9%), nor miscarriage rates (22.1% vs. 21.5%) between the two groups. Women's mean age in group 1 (36.18 ± 3.92) and group 2 (35.88 ± 3.88) was not significantly different (P = .297). A total of 4029 oocytes were vitrified (1980 and 2049 in groups 1 and 2 respectively). A total of 2564 were warmed (1469 and 1095 in groups 1 and 2 respectively). A total of 1386 morphologically eligible oocytes were inseminated by intracytoplasmic sperm injection (792 and 594 respectively, P = .304)., Conclusions: The present study shows that the replacement of the open vitrification system by a closed one has no impact on in vitro and in vivo survival, development, pregnancy and implantation rate. Furthermore, to ensure safety, especially during the current COVID-19 pandemic, the use of the closed device eliminates the potential samples' contamination during vitrification and storage.

Introduction: In Italy, the transport of cryopreserved biological material is controlled by several Decrees (Legislative Decree No. 191/2007 and No. 16/2010 and Health Ministry's Decree of October 10, 2012). Given the nature of their applications, the transport of reproductive cells has peculiar quality and safety requirements that must be applied universally, minimizing the chance of error. To standardize the cross-border shipping procedure to meet the quality, traceability, and safety criteria for cells and tissues, it is appropriate to establish a unified process using the same tools, forms, and communication channels. Methods: A working group has been created by SIERR. This "FOCUS Group" was constituted by representatives from Italian-assisted reproductive technology centers and sperm banks who worked together to define joint procedural steps and create specific forms to support the movement of cryopreserved samples. Results: The FOCUS Group identified the critical steps in the communication procedures between Italian centers and created the related forms: patient authorization, request from the recipient center, critical checks carried out by both sending and recipient centers, start of samples transfer, collection, transport and taking responsibility of the biological material, acknowledgment of samples arrival, and acknowledgement of any adverse event that occurred. Discussion: Indications on shipping between tissue institutions and legal responsibilities are important points and a working protocol with shared transport forms has been defined. Standard Operating Procedures are necessary in light of the increasingly widespread movement of biological samples between the various countries, and represent a valid means of support for the patients who could have a higher awareness of safety and traceability during each stage of gamete transport.

Objective: To investigate spindle behavior during and after slow freezing at room temperature (RT) and vitrification at different temperatures., Design: Randomized, comparative study., Setting: University hospital., Patient(s): Patients undergoing IVF treatment volunteered for the study and donated part of their supernumerary oocytes., Intervention(s): Metaphase II oocytes were divided into group A: slow freezing RT /thawing RT; group B: vitrification RT/warming RT; group C: vitrification RT/warming 37 degrees C; and group D: vitrification 37 degrees C/warming 37 degrees C. Spindle presence was evaluated at each step of the four procedures and in culture., Main Outcome Measure(s): Cumulative spindle recovery rate comparing warming phase of the three vitrification groups and culture phase among the four groups., Result(s): During warming, the three vitrification groups showed a significantly fast spindle recovery rate compared to the thawing of the slow freezing group. A progressively significant fast cumulative recovery rate was observed in the three vitrification groups by increasing the number of phases at physiological temperature (hazard rate = 2.68; 95% confidence interval 1.71-4.02)., Conclusion(s): The present study demonstrates that spindle recovery is faster in vitrification than in slow freezing. These data support a possible protective effect of vitrification/warming at 37 degrees C on the meiotic spindle structure and, therefore, on the subsequent clinical outcome of the procedure, although comparative clinical studies are needed.

Background: The aim of this study was to analyse the relationship between the first polar body (1st PB) morphology and the fertilization rate, cleavage rate, embryo quality, pregnancy and implantation rate., Methods: This was a retrospective study on 167 consecutive cycles undergoing assisted reproduction with ICSI. The 1st PB morphology was evaluated at the moment of ICSI in the 596 injected oocytes and it was coded as intact or fragmented. The fertilization rate, cleavage rate, embryo quality (three grades), pregnancy rate, implantation rate and the time elapsed between oocyte retrieval and ICSI were evaluated. The 1st PB morphology was checked twice (denudation and ICSI) in a random sample of 180 oocytes in order to verify the effect of the in vitro culture., Results: No significant relationship was found between the 1st PB morphology and the fertilization rate (P=0.703), cleavage rate (P=0.055), embryo quality (P=0.673), pregnancy rate (P=0.201) and implantation rate (P=0.511). A significant positive relationship (P=0.006) was found between the frequency of the 1st PB fragmentation and the time elapsed between denudation and ICSI. The pregnancy rate was significantly higher (P=0.008) when oocytes were injected between 5 and 7 h after retrieval rather than earlier or later., Conclusions: Our data suggest that the embryo quality, pregnancy rate and implantation rate are not related to the 1st PB fragmentation. The time which elapses between the oocyte retrieval and ICSI should be maintained at approximately 6 h in order to obtain optimal results.

Since the successful development in the mouse, the oocyte cryopreservation has been applied with varying success to a number of different species including the human. The recently reported successes in terms of pregnancies obtained by human oocyte cryopreservation are encouraging. Several studies typically reported different rates of survival (20-80%), fertilization (30-60%) and cleavage (32-100%). This variability of results throws some doubts on the usefulness of oocyte cryopreservation in IVF treatment cycles. It remains to be determined whether the relatively different success rates reported in literature, mainly in terms of survival rate, are due to methodological differences. We tried to investigate the effect of some factors on the oocyte survival rate after thawing: the presence or absence of cumulus oophorus and the exposure time of the oocytes to cryoprotectant. We suggest that a combination of several factors including both morphological and biophisical ones can affect the oocyte survival rate.

The possibility to employ cryopreservation in Preimplantation Genetic Diagnosis (PGD) should enlarge the opportunities for research and clinical activity. For these purposes, we tried three kinds of approaches on human abnormal embryos: (1) cryopreservation of biopsied embryos; (2) biopsy of thawed embryos; and (3) biopsy of embryos derived from thawed oocytes. Our preliminary results show that: (1) biopsy of thawed embryos is feasible and FISH analysis is possible on both survived and lysed cells; (2) Optimization of freezing/thawing procedures are necessary to obtain better survival rate after thawing of biopsied embryos; (3) Biopsy and FISH are feasible on embryos derived from thawed oocytes and they could be a good way to study the chromosomal arrangement of these poorly investigated embryos.

Human oocyte cryopreservation has met with limited success in terms of both survival and subsequent fertilization. We recently reported the first birth of a healthy female infant after intracytoplasmic sperm injection of cryopreserved oocytes. The current report describes the first pregnancy achieved after intracytoplasmic injection of testicular sperm into cryopreserved human oocytes.

Cryopreservation of human oocytes has been employed with little success in clinical practice, even though it may solve the legal and ethical problems linked to embryo freezing. Various attempts to cryopreserve human oocytes have mostly been unsuccessful, leading to low oocyte survival rates after thawing, and the search for an optimal protocol for oocyte cryopreservation remains elusive. A preliminary study was undertaken to evaluate some of the factors influencing the survival rate of human oocytes and the efficiency of intracytoplasmic sperm injection (ICSI) as an insemination procedure. A total of 38 women with tubal infertility were enrolled in the study. The cryopreservation procedure consisted of a slow freeze-rapid thawing technique using 1,2 propanediol and sucrose as cryoprotectants. The overall oocyte survival rate was approximately 60%. A better survival rate was obtained when the oocytes were cryopreserved in the presence of partially removed cumulus oophorus rather than in the presence of totally enzymatically removed cumulus oophorus. The cryoprotectant concentration and the equilibration time also appear to influence the oocyte survival rate. ICSI may be an efficient method of achieving a satisfactory outcome in terms of fertilization in cryopreserved human oocytes. Embryonic morphological quality does not seem to be compromised by cryopreservation. In conclusion, these data show that cryopreservation may ensure that the integrity of the human oocyte is adequate for normal fertilization and embryo development.

Objective: To describe the first birth achieved after intracytoplasmic sperm injection (ICSI) of cryopreserved human oocytes., Design: Case report., Setting: University of Bologna Hospital, Department of Obstetrics and Gynecology, Reproductive Endocrinology Unit, IVF and Infertility Center., Patient(s): One patient undergoing IVF., Intervention(s): Transvaginal ultrasound-guided oocyte retrieval followed by oocyte freezing. Artificial preparation of the endometrium with E2 and P, oocyte thawing, and ICSI., Result(s): Four of 12 cryopreserved oocytes survived; using ICSI, 2 underwent normal fertilization but only 1 cleaved. One good-quality 4-cell embryo was transferred. A single gestation was confirmed by ultrasound at the 7th week. Amniocentesis was performed at the 16th week and demonstrated a normal female karyotype of 46,XX. After a normal pregnancy, a healthy female infant was born at the 38th week of gestation., Conclusion(s): The combination of ICSI and oocyte cryopreservation is a new tool in assisted reproductive technology.

Transforming growth factor-beta 1 (TGF-beta 1) is a polypeptide involved in a variety of important physiological and pathophysiological processes such as the implantation of the embryo into the endometrium. Many factors seem to be related to this event. TGF-beta 1 is involved in many mechanisms both in endometrial and in embryonic tissues: it induces proliferation and differentiation, it regulates proteolytic activity and it modulates the maternal immune response. This study evaluated the presence of TGF-beta 1 in the endometrium during normal menstrual cycles and in the uterine fluids during induction of ovulation in the framework of an in vitro fertilization program. Immunohistochemistry was used to identify TGF-beta 1 in the endometrium and immunodot-blot to quantitate TGF-beta 1 in the uterine cavity fluid. The study shows that TGF-beta 1 is present in the endometrial tissue and its secretion is modulated during the menstrual cycle, as demonstrated immunohistochemically; its production seems to be controlled by ovarian steroids. In conclusion, TGF-beta 1 influences the growth and differentiation of the embryo, as well as the activation of embryonic proteolytic enzymes, and it modulates the maternal-embryonic immune response. Its variability in the uterine cavity is demonstrated in this study, and the underexpression of TGF-beta 1 in the uterine cavity might be responsible for failed implantation.

Implantation is a crucial step in human reproduction. Disturbances of this process are responsible for pregnancy failure after both in vivo and in vitro fertilization. The endometrium provides the implanting embryo with a unique substratum where the embryo communicates with biochemical signals, attaches itself, penetrates and grows without blood circulation. The highly proliferative phase of the cytotrophoblast, during early human embryogenesis, may be due to endogenous production of growth factors that may establish autocrine/short range paracrine stimulator loops which explain the tumor-like properties of these tissues. Endometrial BM penetration and stroma invasion may be due to the proteolytic capability of the human embryo. It is suggested that collagenase and the urokinase-like plasminogen activator are responsible for this activity. To clarify the molecular mechanisms involved in human embryo implantation several models are suggested: culture of blastocysts, culture of endometrial cells, and endometrial explant co-culture. Human blastocysts cultured with whole perfused human uteri make it possible to recognize some aspects of the entire implantation process and give us the possibility of improving the benefits provided by new technologies in reproductive medicine and reducing embryonic loss at an early stage.

Human uteri were perfused with Krebs-Ringer bicarbonate-glucose buffer with and without estrogens and progesterone for a period of up to 48 hours to preserve a viable organ, which was responsive to hormones. Flow rates of 12 to 35 ml/minute per artery were fully distributed into the organ, with pressure values ranging from 80 to 120 mm Hg. Arteriovenous gradients of oxygen and carbon dioxide tensions as well as the levels of lactate, lactic dehydrogenase, and creatine kinase released in the perfusate, indicators of tissue ischemia or cell necrosis, showed a good preservation of the organ for up to 48 hours. The light- and electron-microscopic examinations of endometrial and myometrial tissues taken before and during perfusion confirmed this result. The extracorporeal perfusion of uteri with buffer containing estrogens plus progesterone exhibited secretive modifications of the proliferative endometrium, thus suggesting the viability of the organ and its responsiveness to sex steroids.

The penetration of luminal epithelium in the uterine cavity represents the crucial event that triggers the failure of embryo implant, thus limiting the possibility of fertility control. The purpose of our study was to implant a human blastocyst, cultured in vitro, into a human uterus extracorporeally perfused with an oxygenated medium. For this purpose, human blastocysts, collected from patients who underwent IVF program because of irreparable tubal infertility, were injected under the luminal epithelium of human perfused uteri. Light and electron microscopy showed that human blastocyst can successfully undergo the stage of implantation and trophoblastic invasion in 52 hours of extracorporeal perfusion.

The methods commonly used to evaluate conjugated steroids require hydrolysis and chromatographic purification. To avoid these steps, a simple method involving selective solid phase extraction and RIA using a highly specific antiserum for estrone sulfate (E1S) has been evolved. A Bond-Elut C2 cartridge was used for solid phase extraction of estrone (E1) and E1S; recoveries were 80 and 90% respectively. The intra- and inter assay precision of the assay at 3 serum levels, were 6.5, 10.4 and 4.4 and 12.7, 13.9 and 7.4% respectively. Accuracy, tested by linearity and recovery tests, was acceptable. A good correlation exists between a conventional enzymatic method and the proposed method. The latter is less time consuming and more reliable, thus providing a rapid assay to evaluate E1 and E1S in the same serum sample.

The present study was undertaken to examine the extractions of estradiol, estrone, and estrone sulfate from the circulation of the human perfused uterus. The differential permeability of endometrial and myometrial vascular beds to estrogens was evaluated in uteri samples obtained during the proliferative and secretive phases of the menstrual cycle. The effects of binding by human serum proteins on estrogen influx into the endometrium and myometrium were also determined by the use of double-isotope, single-injection, timed tissue sampling techniques adapted to the extracorporeal perfusion of human uterus. Tritiated test estrogen was injected into the uterine artery as a mixture with 14C-butanol, a free diffusible reference substance. The influx of 14C-dextran (a membrane-impermeable compound) was used to test the aspecific influx from vasculature to extravascular space. Results show that in the human perfused uterus: (1) membrane permeability plays different roles in estrogen influxes between the endometrium and myometrium; (2) during the proliferative and secretive phase of the menstrual cycle the uterine microvessels are differently permeable to the free plus protein-bound estrogens; and (3) plasma proteins decrease the endometrial and myometrial uptakes of estrogens.