key: cord-0687687-lrsc1plq authors: Stimpfel, Martin; Jancar, Nina; Vrtacnik-Bokal, Eda title: Collecting a semen sample at home for an ART procedure positively affects the blastocyst and embryo utilization rate date: 2020-09-28 journal: Reprod Biomed Online DOI: 10.1016/j.rbmo.2020.09.021 sha: 8eed57c9f7eb0446a9b70012878781d934e06f6c doc_id: 687687 cord_uid: lrsc1plq RESEARCH QUESTION: Does the site of semen collection have any influence on the IVF/ICSI cycle outcome? DESIGN: A retrospective study was carried out at the Department of Human Reproduction, Division of Obstetrics and Gynecology, University Medical Centre Ljubljana. All stimulated and spontaneous IVF/ICSI cycles (with at least one oocyte retrieved) performed in 2019 with fresh ejaculated semen samples were included. The outcome of the ICSI/IVF cycles in terms of oocytes, embryos and pregnancy rates according to the approach of semen sample collection (at home or at clinic) was evaluated. RESULTS: Compared to samples collected at the clinic, semen samples collected at home had significantly higher mean spermatozoa concentrations (60.7 ± 33.0 million/ml vs. 51.9 ± 36.9 million/ml; P=0.001), higher total sperm counts (156.3±113.6 million vs. 138.6±131.4 million; P=0.004) and better motility (59.5% ± 19.6% vs. 55.1% ± 21.9%; P=0.005). The number of retrieved oocytes per cycle was similar (collected at home vs. at clinic; 8.6±7.1 vs. 9.1±6.4; P=0.341). The mean number of embryos was similar between the groups (4.4±4.3 vs. 4.5±3.8; P=0.740), but the blastocyst rate was significantly higher in group where semen was collected at home (52.2% vs. 46.4%; P=0.001). The same was true for the cryopreserved embryo rate (34.7% vs. 30.0%; P=0.003) and embryo utilization rate (56.7% vs. 52.4%; P=0.011). There was no difference in pregnancy rate (collected at home vs. at clinic; 33.8% vs. 34.4%; P=0.888). CONCLUSIONS: Collecting semen at home has a positive effect on sperm quality, blastocyst rate and embryo utilization rate, although it does not affect the pregnancy rate. Coronavirus Disease 2019 is forcing people in professional and private life to adhere to serious preventive measures to minimize the possibility of spreading the virus. The medically assisted reproduction field is no exception, and guidelines from different professional societies were prepared on how to restart treatments or how to change the work to make it as safe as possible. In the guidelines, it was suggested that patients spend the shortest possible time at the clinic, indicating that as much communication as possible should be conducted by telephone, email and so on; these guidelines aimed to not have patients spend unnecessary time in waiting rooms, and another way to achieve this was to have them bring the semen sample collected at home. With regard to semen sample collection, the WHO manual recommends that in normal situations, semen samples should be collected at the clinic near the laboratory to lower the chances of negative external influences on semen quality (World Health Organization, 2010) . The WHO manual suggests that semen should be collected at home only when the patient has difficulties providing the sample with masturbation at the clinic or when there is no appropriate room for semen collection near the laboratory. The use of such an approach should take into consideration that even short exposure of spermatozoa to seminal plasma might negatively influence spermatozoa quality (Björndahl and Kvist, 2003) . The reason for this impact is that seminal plasma is rich in different endogenous compounds, whose concentrations can vary from man to man, and these compounds can have a varied impact on spermatozoa quality (Björndahl and Kvist, 2003) . Another issue is the presence of somatic cells in seminal plasma. These cells are beside abnormal spermatozoa the main source of reactive oxygen species (ROS) (Aitken and Baker, 1995) . Small concentrations of ROS are essential for normal sperm functioning, but when the amount exceeds the capacity of cellular defensive mechanisms, there can be oxidative stress with a serious detrimental effect on sperm DNA (Agarwal et al., 2014) . Another important aspect is the temperature at which the semen is transported to the laboratory. The WHO manual suggests that semen should be kept at temperatures of 20°C to 37°C during transport. It was shown more than 40 years ago that if any delay in semen analysis is expected, the semen should be kept at room temperature (20°C) because the motility and viability decrease significantly faster at 37°C (Appell and Evans, 1977) . Additionally, Appell and Evans (1977) showed that if semen is kept at 4°C, then most of the spermatozoa are immotile after 6 hours, although viability decreases more slowly and remains high after 18 hours. Similarly, Esfandiari et al. confirmed that motility significantly decreases if semen is kept at 4°C but, in contrast, suggested that motility is the highest and ROS levels are the lowest if semen is kept at 37°C (Esfandiari et al., 2002) . If semen is incubated for only 2 hours at 37°C, morphological impairment of sperm nuclei (large vacuoles) can also be observed, while if incubated at 21°C, no such negative effect can be observed (Peer et al., 2007) . Furthermore, the influence of temperature on spermatozoa quality was also studied in native semen as a function of the semen preparation method (density-gradient centrifugation (DGC) versus swim-up technique) and long-term incubation (Thijssen et al., 2014) . It was revealed that after 24 hours of incubation at room temperature (23°C), the motility, viability and morphology were significantly higher than they were in samples incubated at 35°C, but the study could not draw a conclusion regarding which semen preparation method was better in these conditions. Based on available data, there is still no firm conclusion regarding whether collecting semen samples at home has any influence on sperm quality or on IVF/ICSI cycle outcome. At this point, we are utilizing this approach on a daily basis, so our data can be useful in this discussion. A few years ago, we were in situation (clinic renovation) when patients were strongly encouraged to collect semen samples at home and bring them to the laboratory for IVF/ICSI procedures. Because we did not observe any negative influence on the outcome of IVF/ICSI cycles following this approach, we continued this practice. Today, due to the COVID-19 situation, this approach is widely advised, but since the data regarding the success of this approach are limited, we decided to analyse our data and present the outcome of the ICSI/IVF cycles in terms of oocytes, embryos and pregnancy rates according to the semen sample collection approach (at home or at clinic). This retrospective study was carried out at the Department of Human Reproduction, Division of Obstetrics and Gynecology, University Medical Centre Ljubljana. We included all stimulated and spontaneous IVF/ICSI cycles performed from January 2019 to December 2019, where fresh ejaculated semen samples (collected at the clinic or at home) were used for conventional IVF or ICSI procedures and at least one oocyte was retrieved. The data for these cycles were collected from our institutional database of ART procedures. The collection and analysis of these data in anonymized form is allowed by Personal Data Protection Act (Article At our clinic, we offer all patients the option of collecting semen samples at home if they prefer. If the patient collects semen at the clinic, all instructions are given just before collection, but if the patient collects semen at home, all instructions are given in advance in written form, and sterile containers are provided on the day when the follicles are large enough and women get instructions when to administer hCG. In this case, men are advised to ejaculate on that day not to have too long abstinence and are instructed to carry out the semen collection for the IVF/ICSI procedure just before the couple is about to leave home and go to the clinic for oocyte pick-up. This means that ejaculation abstinence is similar for all patients, i.e., approximately 2 days. Usually, the semen is delivered to the IVF lab within 1 to 1.5 hours after collection. Patients are instructed to keep the container with the semen sample close to body temperature during transport. All female patients underwent controlled ovarian hyperstimulation (COH), which was achieved with a GnRH antagonist or a GnRH agonist protocol. In the GnRH antagonist protocol, ovarian stimulation started on day 2 of the cycle. The daily dose of recombinant or urinary gonadotropin was tailored according to the patient's clinical parameters and ranged from doses of 150 IU to 300 IU. Gonadotropin releasing hormone antagonist cetrorelix acetate (Cetrotide; Asta Medica AG. Frankfurt, Germany) or ganirelix (Orgalutran; Organon, Dublin, Ireland) was coadministered subcutaneously daily at a dose of 0.25 mg when the dominant follicle measured ≥ 13 mm in diameter. When at least three follicles measured ≥ I7 mm in diameter, 5.000 IU of human chorionic gonadotropin (hCG) (Pregnyl; Organon, Dublin Ireland) or 6.500 IU (250 mcg) of recombinant hCG alpha (Ovitrelle; Merck Serono Europe, London, England) was administered. Ultrasound-guided transvaginal oocyte retrieval was carried out 36 hours after hCG administration. In the GnRH agonist protocol, daily administration of 0.6 ml of GnRH agonist buserelin acetate (Suprefact; Sanofi-Aventis, Frankfurt, Germany) was started on the 22 nd day of the previous menstrual cycle. After 15 days of treatment or when the endometrium was thin and the ovaries were dormant, a daily dose of gonadotropin was added. The daily dose of recombinant or urinary gonadotropin was tailored according to the patient's clinical parameters and ranged from 200 to 300 IU. When at least three follicles measured ≥ 20 mm in diameter, 5.000 IU of hCG (Pregnyl; Organon, Dublin Ireland) or 6.500 IU (250 mcg) of recombinant hCG alpha (Ovitrelle; Merck Serono Europe, London, England) was administered. Ultrasound-guided transvaginal oocyte retrieval was carried out 36 h after hCG administration. If a semen sample was collected at the clinic, it was incubated for 30 minutes at room temperature to allow liquefaction, and then it was assessed for volume, concentration, and motility. If the sample was collected at home, the assessment was performed immediately after the laboratory received the sample. All assessments were performed on room temperature. The volume was assessed using a graduated disposable pipette. Sperm concentration and total motility were assessed using 20 micron 10x10 grid disposable counting slides (CellVision) according to the manufacturer's instructions. Briefly, 5 microlitres of semen was added to slide, left for 5-10 minutes to stabilize, and then, where possible, at least 200 spermatozoa were counted per slide using phase contrast microscope (400 x magnification). Sperm counting was performed single time and when the count was obviously different from sperm count from previous cycles or from spermiogram, the counting was re-done. The same stands also for sperm motility assessment, which was evaluated from the same sample as sperm counting and calculated as all counted motile spermatozoa divided by count of all spermatozoa. Sperm motility was evaluated under phase contrast microscope (400 x magnification) and spermatozoa were classified only as motile or immotile. The spermatozoa morphology was not evaluated on the day of oocyte pick-up, although it was evaluated before when diagnostic spermiogram was performed. At that point, semen smears were stained using a Papanicolaou method to evaluate the morphology (Tygerberg strict criteria were utilized). Where possible, at least 200 spermatozoa were assessed under 1000 x magnification. Sperm vitality and antisperm antibodies were not examined on the day of oocyte aspiration. In all cases, after initial assessment, the samples were prepared using density gradient centrifugation (100% layer and 40% layer of Pure Sperm (Nidacon)) for 20 minutes at 225 g at room temperature. Then, the 100% layer was washed in 4 ml of Sperm Preparation Medium (Origio), which was followed by centrifugation for 10 minutes at 300 g at room temperature. After centrifugation, the supernatant was discarded, and 0.3-1 ml of Sperm Preparation Medium was added to enable the swim-up. The samples were then put into an incubator at 37°C. After approximately 2 hours of incubation, these samples were either prepared for ICSI or a conventional insemination of cumulus-oocyte complexes was performed. The morning after fertilization methods were carried out, the oocytes were examined for the presence of pronuclei. Normally fertilized oocytes (with 2 PN) were further cultured in continuous culture medium, SAGE 1-Step, or in sequential G-series media (Vitrolife). In cases of sequential media use, embryos were cultured in G-1 Plus medium until the third day, and then they were transferred to G-2 medium. In cases when there were only 1 or 2 embryos, they were transferred into the uterus on the third day of development, at the cleavage stage. In the majority of other cases, when there were more than two embryos, ET was performed on day 5 at the blastocyst stage. ETs were performed using a Guardia Access Embryo Transfer Catheter (Cook Medical), and in all ETs, only 1 or a maximum of 2 embryos were transferred. Supernumerary embryos of appropriate quality were vitrified. When calculating the pregnancy rate, the ETs on day 3 and day 5 were combined and were not analysed separately. To determine the differences between the groups, Pearson's chi-squared and Mann-Whitney U tests were used to analyse the data as appropriate (the normality of data was analysed with Shapiro-Wilk test). P-values of less than 0.05 were recognized as statistically significant. We retrospectively analysed the outcome of IVF/ICSI cycles from 2019 when fresh ejaculated semen collected at the clinic or at home was used to fertilize oocytes. Altogether, there were 1081 cycles with at least one oocyte retrieved. Most of the cycles were stimulated (N=1062), and only 19 were modified natural cycles. The mean age of the women was 35.4 ± 4.7 years, and the mean number of oocytes obtained per cycle was 8.7 ± 7.0. The fertilization rate per all cumulus-oocyte complexes retrieved was 52.3% and 63.7% if the estimated number of MII oocytes was used for calculation. The cleavage rate of fertilized oocytes was 97.9%, which resulted in 4.5 ± 4.2 embryos per cycle. Some embryos were transferred at the cleavage stage on day 3, but most of them were cultured until day 5/6, and 50.9% of them developed to blastocysts. When the utilization rate of embryos (defined as the proportion of all transferred and cryopreserved embryos per number of all obtained embryos (regardless of the day of transfer or of cryopreservation)) was calculated, 55.8% of them were usable. Embryo transfer (ET) was performed in 78.2% of cycles, while the rest of the cycles were either freeze-all (11.5%), or no appropriate embryo for ET or cryopreservation was obtained in the cycle (10.3%). The mean number of transferred embryos was 1.3±0.4, and the pregnancy rate per ET was 34.0%. Pregnancy was defined as a positive βHCG test 15 days after ET. For the analysis of cycles according to the site of sperm sample collection, there were 837 cycles where semen samples were collected at home and 244 cycles where semen samples were collected at the clinic. The data revealed that samples collected at home had significantly higher mean spermatozoa concentrations (60.7 ± 33.0 million/ml vs. 51.9 ± 36.9 million/ml, respectively; P=0.001), total sperm counts (156.3 ± 113.6 million vs 138.6±131.4 million, respectively; P=0.004) and better motility (59.5% ± 19.6% vs. 55.1% ± 21.9%, respectively; P=0.005), but the semen volume and morphology did not differ (Table 1) . When the laboratory outcome of cycles was analysed ( Similarly, the cryopreserved embryo rate (34.7% vs. 30.0%; P=0.003) and embryo utilization rate (56.7% vs. 52.4%; P=0.011) were significantly higher in the group of patients in which semen samples were collected at home. Despite these differences, there was no difference in pregnancy rate (collected at home vs. at clinic; 33.8% vs. 34.4%; P=0.888). Additional analysis of pregnancy rates also accounted for the methods used to fertilize oocytes (conventional IVF vs. ICSI) and did not reveal any significant difference (Table 3) . To further reveal what could be the cause of the observed significant differences, we analysed different subgroups of patients. To determine whether a significantly higher proportion of immature oocytes in the group of patients who collected semen at the clinic influenced preimplantation embryo development, we analysed only cycles where the number of retrieved oocytes was greater than the overall mean number, and we excluded patients who had a proportion of immature oocytes that was higher than the average overall proportion from this group. This means that we included only cycles with 9 or more retrieved oocytes and with a proportion of immature oocytes that was less than 17.8%. The results are presented in Table 4 . Briefly, the mean number of oocytes and embryos was similar between groups, as was the proportion of immature oocytes and the pregnancy rate. For the main categories of interest (blastocyst rate, utilization rate and embryo cryopreservation rate), the differences remained significantly different, with higher rates of each category in group that performed semen collection at home. The results of this study indicate that IVF laboratory outcomes are at least comparable, if not better, if semen samples for IVF/ICSI procedures are collected at home and not at the clinic. Our data suggest that fertilization and embryo cleavage rates are the same regardless of the site of semen sample collection, but the blastocyst rate and embryo utilization rate are improved if a semen sample is collected at home. Because we analysed only data for the previous year (2019), we could not determine if there was any influence on the abortion and birth rate, although the current data suggest that pregnancy rates were similar between the compared groups. Because there are several possible external influences that can negatively affect the quality of spermatozoa and consequently the outcome of the IVF/ICSI procedure, our results are somewhat surprising. Furthermore, we could not find any study addressing the question if there is any influence on the outcome of IVF/ICSI procedure if semen sample is collected at home. On the other hand, two studies explored whether there was any influence on intrauterine insemination (IUI) outcome regarding the location of semen collection (Song et al., 2007; Yavas and Selub, 2004) . Yavas and Selub (2004) concluded that in their study, semen collection at the clinic led to a better pregnancy rate, but this occurred only when women were stimulated with hMG. When women were stimulated with clomiphene citrate, there was no difference in pregnancy rates based on the site of semen collection. Furthermore, they suggested that if the start of semen preparation is delayed from 30 minutes to 1 hour or more, then the pregnancy rate can be impaired. They suggested that the pregnancy rate can also be impaired if IUI is performed more than 90 minutes after the semen is prepared for treatment. On the other hand, a study by Song et al. (2007) did not find any difference in the ongoing pregnancy rate when patients collecting semen at the clinic were compared with those who collected semen at home. There was also no difference in semen parameters, although the time from semen collection to the IUI procedure was significantly longer in patients who performed semen collection at home. We do not have data for our patients regarding the time from semen collection at home to semen processing. We can only estimate that is probably longer than the time lapse for patients who performed semen collection at the clinic; nevertheless, we observed that semen parameters, in terms of concentration and motility, were better when semen was collected at home. Our observation that semen parameters were better when semen was collected at home is consistent with the results of a study by Elzanaty and Malm (2008) . Their study showed that home-collected semen samples had a significantly higher spermatozoa concentration, total sperm count, and motility, while there was no difference in the morphology or the concentration of biochemical substances (neutral alpha-glucosidase, prostate-specific antigen, zinc, and fructose). Furthermore, significantly fewer home-collected samples had abnormal sperm concentration or abnormal rapid progressive motility. While this study showed a positive effect on semen quality when samples were collected at home, a study by Shetty Licht (2008) did not find any statistically significant difference in sperm count, sperm motility, total count, or total motile count between those samples collected at home and those collected at clinic. The reason for such observation may be derived from the study design because the patients could decide for themselves if they preferred to perform the collection at home or at the clinic. In this way, the semen was collected at the clinic only by patients who were comfortable with this option, while the patients who could be under elevated stress performed semen collection at home. The first studies evaluating the relationship between psychological stress and semen quality suggested that stress negatively affects the quality of semen in patients who are involved in IVF treatment (Clarke, 1999; Harrison et al., 1987; Ragni and Caccamo, 1992) , but later, this conclusion was challenged (Nouri et al., 2014) . Nouri et al. (2014) suggested that semen quality indeed declines during IVF, but this is probably not due to subjective stress. Despite this, they showed that in couples with subjective male stress, there were more poor responders, more miscarriages and fewer live births. Haidl (2014) further suggested that poorer outcome of IVF treatment may occur due to acute stress in males and because functional parameters of semen quality could be impaired in addition to basic parameters. For instance, (Vellani et al., 2013) showed that male stress was associated with increased sperm DNA fragmentation in IVF patients. An additional factor that could potentially influence the quality of semen is the period of sexual abstinence. The WHO recommends abstinence of 2-7 days (World Health Organization, 2010), but the optimal sexual abstinence period is still debatable. Some semen quality parameters can improve with longer periods, but others improve with shorter periods (Hanson et al., 2018) . Meta-analysis by Hanson et al. (2018) suggests that longer abstinence increases semen volume and sperm count, but motility, morphology, and DNA fragmentation seem to improve with shorter periods of abstinence. These studies mostly focused only on sperm quality parameters, so there is not much data in the literature on how the sexual abstinence period influences the outcome of an ART cycle. Periyasamy et al. (2017) showed that clinical pregnancy rates were significantly higher if the abstinence period was less than 8 days; furthermore, the positive effect of a shorter abstinence period was also revealed in a higher live-birth rate per ET. There are other studies showing that a shorter abstinence period positively influenced ART cycle outcomes. Jurema et al. (2005) suggested that pregnancy rates in IUI are improved if abstinence is less than 4 days (Jurema et al., 2005) , while Colturato et al. (2007) further suggested that in ICSI cycles, the best quality of embryos and the highest pregnancy rate are achieved after abstinence period of 1 day; a similar result was confirmed by Borges et al. (2019) . Furthermore, recurrent ejaculation for 4 days with a final abstinence period of 12 hours was shown to improve the pregnancy rate in ICSI cycles (Sánchez-Martín et al., 2013) . Because we want to avoid a long sexual abstinence period in our IVF programme, which can potentially impair the quality of semen, we aimed to ''synchronize'' the abstinence period with the planned oocyte pick-up. This can be optimally carried out by advising the patients to ejaculate on the day when the follicles are large enough to administer hCG, meaning that usually approximately 2 days pass before men collect semen for the IVF/ICSI procedure. Therefore, we can say that the abstinence period is most likely similar for all patients and does not influence the results of our analysis and this is also one of the strengths of our study. Another strength is the large number of included cycles, which are distributed throughout the entire year. In this way, the influence of external factors (e.g., temperature) or other factors (fluctuations in the outcome of IVF/ICSI cycles) is minimized. On the other hand, because this is a retrospective study and we included all patients regardless of their diagnosis, there could be a bias to our results. Another bias could be the analysis of all cycles and not only the first cycles. The prognosis is better for the first cycle than subsequent cycles and unbalanced distribution of first-cycle patients between the two arms may lead to bias. Therefore, we suggest that a prospective randomized trial taking all these limitations into account should be performed in the future. In conclusion, in this study, we retrospectively analysed whether collecting semen samples at home influences sperm quality or IVF/ICSI cycle outcome in terms of oocytes, embryos and pregnancy rates. The results revealed that collecting semen at home has a positive effect on sperm quality (sperm concentration, total sperm count and motility were all higher), blastocyst rate and embryo utilization rate. The number of transferred embryos was similar in both groups, but the proportion of cryopreserved embryos was significantly higher in the home-collected semen group; therefore, we propose that this could potentially lead to a higher cumulative pregnancy/birth rate in this group. Based on these data, we suggest that collecting semen at home for IVF/ICSI procedures is safe and positively influences treatment outcomes. The data for all parameters except morphology are for assessments performed on the day of oocyte pick-up. On the day of oocyte pick-up, the morphology was not evaluated, although it was evaluated before when a diagnostic spermiogram was performed. The observed significant differences are marked with an asterisk (P value < 0.05). Andrology: Seminal leukocytes: passengers, terrorists or good Samaritans? The Effect Of Temperature On Sperm Motility And Viability Sequence of ejaculation affects the spermatozoon as a carrier and its message Revisiting the impact of ejaculatory abstinence on semen quality and intracytoplasmic sperm injection outcomes Relationship between psychological stress and semen quality among invitro fertilization patients Influence of sexual abstinence length on sperm parameters and on IVF outcomes in ICSI assisted treatment cycles Comparison of semen parameters in samples collected by masturbation at a clinic and at home Effects of temperature on sperm motion characteristics and reactive oxygen species. International journal of fertility and women's medicine Commentary on ′Decline of semen quality during IVF is not associated with subjective male stress′ The impact of ejaculatory abstinence on semen analysis parameters: a systematic review Stress and semen quality in an in vitro fertilization program Effect of ejaculatory abstinence period on the pregnancy rate after intrauterine insemination Decline of semen quality during IVF is not associated with subjective male stress Is fine morphology of the human sperm nuclei affected by in vitro incubation at 37°C? Negative effect of stress of in vitro fertilization program on quality of semen Increased pregnancy after reduced male abstinence. Systems biology in reproductive medicine Site of semen collection and its effect on semen analysis parameters Location of semen collection and time interval from collection to use for intrauterine insemination Influence of temperature and sperm preparation on the quality of spermatozoa Association of state and trait anxiety to semen quality of in vitro fertilization patients: a controlled study WHO laboratory manual for the examination and processing of human semen: Fifth edition. World Health Organization Intrauterine insemination (IUI) pregnancy outcome is enhanced by shorter intervals from semen collection to sperm wash, from sperm wash to IUI time The authors would also like to thank all gynaecologists, clinical embryologists, medical nurses, and other staff at the Department of Human Reproduction, Division of Gynaecology, University Medical Centre Ljubljana, for their support. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.