Abstract Study question Can the use of Theophylline recover motility of frozen surgically retrieved sperms in case of absence of motility after thawing? Summary answer Theophylline allows to recover motility of thawed surgically retrieved sperms. The utilization of sperms with or without pharmacological activation gives comparable clinical outcomes. What is known already Testicular sperm motility is usually poor. A method is needed to detect viable sperm for ICSI when motility is totally absent after freezing/thawing. Hypo-osmotic swelling test, mechanical touch technique, laser-assisted immotile sperm selection, birefringence-polarization microscopy and exposure to pharmacological stimulation are techniques used for this purpose. Among pharmacological agents Dimethylxanthine Theophylline is a phosphodiesterase inibitor that improves sperm motility by promoting an increase in intracellular cyclic AMP levels. Few studies report that it is efficient for recovery of sperm motility in cases of thawed testicular and retrograde ejaculation samples improving reproductive outcomes. Study design, size, duration Retrospective analysis of sixty frozen surgical sperm cycles (45 patients) utilized from February 2018 to November 2020. After thawing, samples were divided in two Groups according to motility recovery. Group A: presence of motility, Group B: absence of motility. Group B was treated with Theophilline and motility was re-assessed after incubation. Activated sperms were utilized for ICSI when available. Sperm motility recovery, fertilization, pregnancy rate/transfer, implantation and miscarriage rate were evaluated in both Groups. Participants/materials, setting, methods Surgical specimens were treated and concentrated in SpermRinse™ Medium (Vitrolife) and then cryopreservated in nitrogen vapor in TEST Yolk Buffer (Irvine Scientific). After thawing, only samples with no motility recovery were treated with a brief incubation in Theophylline (GM501 SpermMobil, Gynemed) and washed in Polyvinylpyrrolidone (ICSITM Vitrolife) before injection. ICSI was performed in all cases approximately 4–5 hours after sperm thawing. After fertilization check, transfer was scheduled in day 2. Main results and the role of chance Women’s age Group A (34,39±2,29 M±SD) and group B (35,87±4,34 M±SD) and men’s age Group A (37,31±5,12 M±SD) and group B (40,89±8.15 M±SD) were not significantly different (P= .328 and P=.218) respectively. Group A: 13/60 cycles (21.7%) (9 patients). Pre freezing and post thawing total motility percentage were 34.0±19.0 (M±SD) and 13.5±15.6 (M±SD) respectively (39.8% recovery). Group B: 47/60 cycles (78.3%) (36 patients). Pre freezing total motility percentage was 5.3±8.5 (M±SD) and no motility was recovered post thawing (0%). After treatment with Theophylline total motility was 1.8±1.8 (M±SD) (33.5% recovery). Motile sperms were utilized in all cases except from two in the Group B. Number of injected oocytes was 2.8±1.1 (M±SD) in Group A and 4.3±3.1 (M±SD) in Group B (P=.004) respectively. Fertilisation rate (63.1% and 45.4%, P=.066), Number of embryos transferred (1.8±0.7 M±SD and 1.6±0.7 M±SD, P=.271), Pregnancy rate/Transfer (54.5% and 37.1%, P=.502), Implantation rate (30.0% and 27.8%, P=.919) and Miscarriage rate (33.3% and 30.7%, P=.675) were not statistically significant between Group A and B respectively. In the two cases of group B injected with immotile sperm, fertilization rate was 0% (0/3) and 50% (2/4). Limitations, reasons for caution A larger study is needed to investigate the recovery of sperms motility (and/or their activation) and clinical outcomes, in particular referring to the origin of sampling (epididymal aspirate and testicular tissue) and type of azoospermia (obstructive and non-obstructive). Wider implications of the findings: Theophylline is an effective tool for sperm motility recovery after thawing allowing to inject viable sperm and facilitating laboratory handling. Trial registration number Not applicable

Abstract Study question Is Sars-Cov–2 present in the follicular fluid of infertile patients? Summary answer In the experience of the Infertility and IVF Unit, University Of Bologna, Italy, RNA of SARS-Cov–2 was not detected in the follicular fluid. What is known already Data on the risk of virus presence in reproductive cells and transmissibility in IVF procedures are very limited. In literature only one study reports the detection of SARS-Cov–2 viral RNA in oocytes of PCR positive women. Research of RNA in follicular fluid could be a marker able to indicate whether to continue IVF treatments in the case of swab-positive patients. Study design, size, duration Prospective study performed at Infertility and IVF Unit, Sant’Orsola University Hospital, University of Bologna, Italy, from March 2020 to January 2021. 451 IVF cycles were performed on 902 patients. In addition 59 cycles of oocyte cryopreservation were also performed to fertility preservation in oncological patients. In all positive swab patients was analyzed the follicular fluid for RNA virus detection. Participants/materials, setting, methods 961 patients underwent telephone triage before going to the IVF Center to identify subjects with suspected or confirmed infection. Body temperature was measured on all patients before entering the IVF Center. All patients were subjected to real-time analysis (RT PCR) of pharyngeal swab samples 48 hours before transvaginal ultrasound-guided oocyte retrieval. In case of positive swab, PCR was performed on follicular fluid. Main results and the role of chance In our population of infertile patients the incidence of SARS COV–2 infection positivity was 0.4% (4/961). No IVF treatments were suspended. The oocytes of the 4 women with positive swab were cryopreserved using closed devices stored in a special dedicated cryogenic container. No viral RNA was detected in the follicular fluid. Limitations, reasons for caution there are no limitations to the study. Wider implications of the findings: The absence of SARS-COV–2 RNA in the follicular fluid is a reassuring result in the storage and future use of oocytes. Trial registration number Not applicable

the study aims to determine the impact of BRCA1 and BRCA2 germline mutations on fertility preservation outcomes. retrospective study. 65 breast cancer patients between 18 and 40 years old undergoing a fertility preservation treatment at the University of Bologna IVF Center from 1997 to 2017 were considered. 21 patients had not performed the test, therefore were excluded from the study. Patients were divided in four group: Group A was composed by 9 BRCA 1-positive patients, Group B with 11 BRCA 2-positive patients, Group C with 24 breast cancer BRCA-negative patients and Group D, control group composed by 181 women with a mean age of 32.4±2.8 years who underwent and ovarian stimulation due to male factor. All patients were stimulated with FSH-r and GnRH agonist or antagonist. In breast cancer patients with hormone-sensitive neoplasm, aromatase inhibitor was used. General characteristic and ovarian stimulation aspects were analyzed. T-student test was used to analyze the data. All the results are shown in Tab. 1. Group A showed significant lower AMH levels compared to Group C and D. Furthermore patients in group A showed significant lower AFC compared to the control group (Group D). Maximal estradiol serum levels were not considered due to the Letrozole use. BRCA 1+ patients showed a significant lower rate of mature oocytes (MII) compared to Group D (4.1±3.43 vs 6.3±3.4 p<0,05). No significant differences were found between the groups considering basal FSH levels, duration of stimulation, number of follicles developed and number of oocytes retrieved. The study showed that BRCA 1 patients had a higher risk of premature ovarian failure confirmed by a diminished ovarian reserve (lower AMH and AFC) and a lower number of cryopreserved oocytes. Therefore women should be properly counseled about this risk and oocytes cryopreservation option should be suggested considering the recommendation of a prophylactic bilateral salpingo-oophorectomy at 40 years old for both BRCA 1 and 2 women.Tabled 1ResultsGROUP AGROUP BGROUP CGROUP DAGE31.3±3.2732.63±4.2232.87±4.3832.4±2.8AMH1.85±1.344.36±5.314.54±4.053.8±2.5BASAL FSH6.7±1.25.85±1.916.57±2.567.1±2.2AFC10±1.411.45±0.9312.65±1.2612.48±1.36DAYS OF STIMULATION10.66±2.4910.18±2.7510.79±2.6511.3±3.25TOTAL DOSE OF GONADOTROPINS2012.5±1298.221670.45± 827.782106.25± 1336.141597±709FOLLICLES >16MM13.2±4.5813.54±5.3113.08±5.6712.26±5.04N. OOCYTES RETRIEVED7.3±4.659.90±6.509.12±6.068.8±4.3N. CRYOPRESERVED OOCYTE4.1±3.437.27±5.747.15±4.416.3±3.4 Open table in a new tab

Changes in global temperature are believed to influence fertility. Sperm counts are lowest in summer months and highest in winter. The decrease of birth rate in the last century in all industrialized countries is related to exponential increase of Earth temperature. Very little is kwon about the relationship between global temperature and oocyte development. Several studies in animals showed that IVF achieves better outcomes in cold temperature mainly because hot climates promote oxidative stress which is directly involved in energetic changes and in cellular metabolic homeostasis. The aim of the study is to determine whether seasonal changes of temperature affect follicular recruitment, number of oocytes retrieved and pregnancies achieved in assisted reproduction technology (ART). IVF cycles performed in Our Centre from 2010 to 2015 were analyzed and divided in two groups according to the month of execution: cold month from December to February (with a mean temperature of 4°C, 39, 2°F) and hot months from june to july (with a mean temperature of 24,4°C, 75, 9°F). All patients underwent ovulation induction with recombinant FSH. 1645 cycles were studied, of which 841 in cold months and 804 in hot months. The two groups showed no differences in women age (36 years in cold months vs 36,3 in hot months), ovarian reserve (average FSH and AMH respectively: 8,5 UI/L in cold months vs 8,7 UI/L in hot months and 2,2 ng/ml in cold months and 2,3 ng/ml in hot months) and average number of oocytes retrieved (6 in cold months vs 5,9 in hot months). The study showed no differences also in fertilization rate (75,7% in cold months vs 75,1% in hot months) and pregnancy rate per transfer (29,3% in cold months vs 30,2% in hot months). The abortion rate was respectively 21,7% and 25% in cold and hot months. Our study showed no significant differences in number of oocytes retrieved and pregnancies achieved. Literature suggests that temperature changes can affect natural fertility, but until now no clinical data confirm this influence in IVF treatment.

Anti-neoplastic treatments have significantly increased the survival of cancer patients, but female patients risk premature menopause. Oocyte cryopreservation has been proposed as a fertility-saving option. This report describes the first live birth achieved with autologous cryopreserved oocytes in an ovariectomized borderline cancer patient. A patient with a borderline ovarian tumour asked for oocyte cryopreservation after a right adnexectomy. Ovulation induction resulted in the retrieval and cryopreservation of seven mature oocytes. Thirty-nine months after a left ovariectomy, the patient asked for oocyte thawing and embryo transfer. Endometrial growth was induced using hormone replacement treatment. Three of the seven cryopreserved oocytes were thawed; they survived and, after insemination, normal fertilization took place. Three embryos were transferred into the patient's uterus. A twin pregnancy was achieved with the birth of two healthy females. Oocyte cryopreservation may be a reliable option for preserving fertility in young cancer patients who risk premature menopause due to surgery, chemotherapy or radiotherapy.

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.

Objective: The problem of supernumerary embryos in assisted reproduction techniques (ART)is still unsolved. Embryo cryopreservation often only postpones the problem and, in addition, it is not allowed in all Countries. Oocytes cryopreservation may be an alternative especially now that a certain efficiency of this technique has been demostrated. Insemination of a limited number of oocytes and storage of supernumerary eggs would have an ethical advantage without reducing the cycle efficiency. The aim of this study is to compare the effects of inseminating 4 or more than 4 eggs in terms of fertilization and pregnancy rates and the results of excess oocytes or embryo cryopreservation.Design: Evalutation of the efficiency of inseminating a limited number of oocytes and storing the excess eggs.Materials/Methods: Superovulation was induced with the association of recombinant FSH (Gonal-F) and GnRH agonist (Enantone) and transvaginal oocytes pick-up was performed in 78 patients undergoing ART. In 41 cycles (group 1)no more than 4 oocytes were inseminated and excess eggs were frozen while in 37 cycles (group 2) all the oocytes were insemineted and excess embryos were frozen. ICSI and standard culture procedures were used and embryo transfers were done on day 2.Results: In group 1, 360 eggs were retrieved, 206 were frozen, 124 mature oocytes were inseminated. The fertilization rate was 76%. A mean number of 2.7 ± 1.0 embryos were transferred per cycle. In group 2, 433 oocytes were collected and 346 mature eggs were inseminated with a fertilization rate of 69%. 2.6 ± 1.0 embryos per cycle were transferred and 3.5 ± 0.5 ± per cicle were frozen. Embryo quality was similar in the two groups. Pregnancy rate per transfer was 24.2% and 26.4% in group 1 and group 2 rispectively. Subsequent pregnancy rate in 19 thawing cycles of frozen oocytes were 17.2% and 18.7% in 23 thawing cycles of cryopreserved embryos.Conclusions: Comparison between elective insemination of a limited number oocytes or insemination of all eggs retrieved documented a similar fertilization rate, embryo quality and pregnancy rate. Pregnancy rate from thawed oocytes and thawed embryos are also similar. These data documents that storing excess eggs may be an efficient alternative to embryo cryopreservation with ethical and legal advantages. Objective: The problem of supernumerary embryos in assisted reproduction techniques (ART)is still unsolved. Embryo cryopreservation often only postpones the problem and, in addition, it is not allowed in all Countries. Oocytes cryopreservation may be an alternative especially now that a certain efficiency of this technique has been demostrated. Insemination of a limited number of oocytes and storage of supernumerary eggs would have an ethical advantage without reducing the cycle efficiency. The aim of this study is to compare the effects of inseminating 4 or more than 4 eggs in terms of fertilization and pregnancy rates and the results of excess oocytes or embryo cryopreservation. Design: Evalutation of the efficiency of inseminating a limited number of oocytes and storing the excess eggs. Materials/Methods: Superovulation was induced with the association of recombinant FSH (Gonal-F) and GnRH agonist (Enantone) and transvaginal oocytes pick-up was performed in 78 patients undergoing ART. In 41 cycles (group 1)no more than 4 oocytes were inseminated and excess eggs were frozen while in 37 cycles (group 2) all the oocytes were insemineted and excess embryos were frozen. ICSI and standard culture procedures were used and embryo transfers were done on day 2. Results: In group 1, 360 eggs were retrieved, 206 were frozen, 124 mature oocytes were inseminated. The fertilization rate was 76%. A mean number of 2.7 ± 1.0 embryos were transferred per cycle. In group 2, 433 oocytes were collected and 346 mature eggs were inseminated with a fertilization rate of 69%. 2.6 ± 1.0 embryos per cycle were transferred and 3.5 ± 0.5 ± per cicle were frozen. Embryo quality was similar in the two groups. Pregnancy rate per transfer was 24.2% and 26.4% in group 1 and group 2 rispectively. Subsequent pregnancy rate in 19 thawing cycles of frozen oocytes were 17.2% and 18.7% in 23 thawing cycles of cryopreserved embryos. Conclusions: Comparison between elective insemination of a limited number oocytes or insemination of all eggs retrieved documented a similar fertilization rate, embryo quality and pregnancy rate. Pregnancy rate from thawed oocytes and thawed embryos are also similar. These data documents that storing excess eggs may be an efficient alternative to embryo cryopreservation with ethical and legal advantages.

Objective: Among the advantages of oocytes cryopreservation are the lack of ethical and legal problems which are associated with embryo storage, and major benefits for patients who risk loss of ovarian function. However, human cryopreservation has met with limited success both in terms of survival and subsequent fertilization. The advantage of intracytoplasmatic sperm injection (ICSI) of cryopreserved oocytes has been described recently in terms of higher fertilization and cleavage rate. We reported the first birth of a healthy female after ICSI of cryopreserved human oocytes. The present report describes four births achieved after intracytoplasmic sperm injection of cryopreserved human oocytes with cryopreserved human sperms. Design: N/A. Materials/Methods: Four women with tubal infertility were enrolled in our assisted reproduction program. Multifollicular development was induced with a combination of a GnRH analogue and menotropins. Ovulation was induced with 10000 IU of hCG when 15 follicles ≥16 mm in mean diameter were developed. A transvaginal ultrasound guided oocyte pick-up was performed 34 hours later were retrieved. Since the patients partners failed to produce the semen sample, the patients was included in our oocytes freezing program. Oocytes were cryopreserved with the freezing protocol previously reported while to avoid the risk of further failures of semen production, a semen sample was frozen in Freezing Medium diluted 1:1. Some months after, the patients underwent to a hormonal replacement therapy in order to improve endometrial growth. In the presence of adequate stimulation (8 mm) both oocytes and semen were thawed; sperm selection was done by minipercoll technique and intracytoplasmic sperm injection (ICSI) technique was performed. Results: Survival rate of thawed oocytes was 64%. The fertilization rate was 59%. On day 3 embryos was transferred. Healthy gestations were confirmed by ultrasound examination on day 35 with the presence of embryonic cardiac activity. The amniocentesis performed at 16th week of gestation revealed a normal karyotype. Four normal healthy baby were born. Conclusions: The combination of oocytes cryopreservation and ICSI described by our group has greatly improved the results rate with thawed oocytes. The presents case document can be safely used together to obtain healthy offspring.

Oocyte cryopreservation is a viable solution for the ethical problems related to embryo storage, and the only available technique for preservation of fertility in women who have to undergo chemo- or radiotherapy. The main problems with oocyte cryopreservation are concerned with the survival rate and the fertilization rate. Recently the introduction of the intracytoplasmic sperm injection (ICSI) led to an increase in the fertilization rate. The success achieved with the first case treated encouraged us to set up a clinical trial on human oocyte cryopreservation. In the first stage of the study, 23 women with tubal infertility were enrolled. Superovulation was induced and 375 oocytes were retrieved; of these 338 oocytes were frozen. The survival rate was 59.5% and was independant of the duration of cryopreservation or the presence of cumulus. The normal fertilization rate was 64.4%, and only 7.5% of fertilizations were abnormal. A total of 90.8% of fertilized oocytes cleaved. A mean of 3.1+/-1.3 embryos per patient were transferred. Three pregnancies were achieved. In the second stage of our investigation, more patients were enrolled and similar results were observed. Sixteen pregnancies were achieved. A further stage of the investigation involved the fertilization of frozen oocytes with frozen sperm and even these resulted in a pregnancy. Our study demonstrated that pregnancies can also be achieved when frozen eggs are fertilized by testicular and epididymal sperm. As a consequence of the success of our investigations, a program of oocyte cryopreservation for oncological patients has been initiated in our centre. In our opinion, oocyte cryopreservation is, at present, a safe and efficient technique as documented by the birth of several healthy children.

In 1997, the ESHRE PGD Consortium was formed as part of the ESHRE Special Interest Group on Reproductive Genetics, in order to undertake a long-term study of the efficacy and clinical outcome of preimplantation genetic diagnosis (PGD). In December 1999, the first PGD Consortium report was published discussing referrals of 323 couples, 392 PGD cycles and 82 pregnancies and 79 children born. In the second round of data collection, contributing centres were asked to send in data from their PGD activities before January 1997, as well as from 1st October 1998 until 1st May 2000, in order to have as complete as possible an overview of PGD practices in these centres. A further 563 referrals were sent in as well as 926 PGD cycles, and data on 89 pregnancies (including seven pregnancies ongoing from the previous group) and 83 children were collected. This has led to a considerable amount of cumulative data being acquired: over a period of 7 years (the oldest PGD cycle reported dates from 1994), referral data on 886 couples, cycle data on 1318 PGD cycles and data on 163 pregnancies and 162 babies were collected. In all, these data are encouraging: they show first, that the practice of PGD is becoming more and more established, and an increasing number of different applications is emerging; and second, that collecting these data is worthwhile, as they will be a valuable source of information for all those involved, e.g. in counselling patients and interacting with governmental bodies.

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.