Early and late paternal contribution to cell division of embryos in a time-lapse imaging incubation system

The objective of this study was to investigate the impact of male age, semen quality and days of ejaculatory abstinence on embryo morphokinetics. A total of 1,220 zygotes obtained from 139 couples in a private in vitro fertilisation centre were analysed. The timing of specific events from the point of insemination, such as timings to pronuclei appearance and fading, to two, three, four, five, six, seven and eight cells and to blastulation were recorded. Multivariate linear regression analysis was used to evaluate the influence of paternal factors on embryo morphokinetic events. Paternal age was positively correlated with delayed cell cleavage and blastulation, and negatively associated with implantation rate, and clinical pregnancy and live–birth chances. The ejaculatory abstinence was inversely correlated with the implantation rate. Inverse relationships were observed between semen parameters (sperm count, progressive sperm motility, total motile sperm count and morphology) and the timing of specific events during embryo development. Sperm morphology was also positively associated with implantation rate and pregnancy and live–birth chances. Increased paternal age and ejaculatory abstinence, and poor semen quality correlate with delayed cell cleavage and blastulation and negatively impact intracytoplasmic sperm injection outcomes.     

ICSI outcome in patients with high DNA fragmentation: Testicular versus ejaculated spermatozoa

Sperm DNA fragmentation (SDF) has emerged as an important biomarker in the assessment of male fertility potential with contradictory results regarding its effect on ICSI. The aim of this study was to evaluate intracytoplasmic sperm injection (ICSI) outcomes in male patients with high SDF using testicular versus ejaculated spermatozoa. This is a prospective study on 36 men with high‐SDF levels who had a previous ICSI cycle from their ejaculates. A subsequent ICSI cycle was performed using spermatozoa retrieved through testicular sperm aspiration. Results of the prior ejaculate ICSI were compared with those of the TESA‐ICSI. The mean (SD) SDF level was 56.36% (15.3%). Overall, there was no difference in the fertilization rate and embryo grading using ejaculate and testicular spermatozoa (46.4% vs. 47.8%, 50.2% vs. 53.4% respectively). However, clinical pregnancy was significantly higher in TESA group compared to ejaculated group (38.89% [14 of 36] vs. 13.8% [five of 36]). Moreover, 17 live births were documented in TESA group, and only three live births were documented in ejaculate group (p < .0001). We concluded that the use of testicular spermatozoa for ICSI significantly increases clinical pregnancy rate as well as live‐birth rate in patients with high SDF.     

Clinical outcome after insemination with donor sperm in patients with poor results in ICSI cycles

The introduction of intracytoplasmic sperm injection (ICSI) provided an effective treatment for infertile couples whose infertility was attributed to male factors. However, some of them face poor results after ICSI and subsequently use artificial insemination with donor sperm (AID). Only a few studies have reported on the clinical outcome of AID cycles after previous failed ICSI cycles, with contrasting results. The results reported here involve a cohort of 47 couples undertaking 175 AID cycles after 120 failed ICSI cycles for various reasons. Couples were allocated to two groups according to the availability of top quality embryos (TQE) in ICSI cycles. In our series, AID was successful for couples with and without TQE previously transferred in ICSI cycles, the live birth rate (LBR) per cycle being 20.0% and 13.3%, respectively. However, couples with TQE tended to succeed more rapidly than couples with poor quality embryos, with a higher cumulative LBR (68.0% versus 54.5%, respectively). These findings demonstrate that even couples with a history of unsuccessful ICSI cycles because of poor embryo quality are able to achieve high LBR after AID cycles. However, such couples have a lower cumulative LBR and are required to be more patient to achieve parenthood.     

Prognostic value of male diagnostic profiles in intracytoplasmic sperm injection (ICSI)

Possible correlations between male hormone and semen parameters with pregnancy and oocyte fertilization rates following intracytoplasmic sperm injection (ICSI) were investigated. The study is based on 290 couples who underwent ICSI therapy for the first time. The parameters evaluated were male age, serum levels of follicle stimulating hormone (FSH) and testosterone, sperm concentration, sperm motility, normal sperm morphology, index of teratozoospermia (TZI) and sperm vitality. A marginal, barely significant association was found between the fertilization rate and serum FSH levels in the male partner ( p =  0.046). There was no relevant association between male parameters and pregnancy rates. The study confirms that male hormonal and semen parameters are of low prognostic value for the outcome of ICSI.     

A comprehensive work up for an asthenozoospermic man with repeated intracytoplasmic sperm injection (ICSI) failure

Infertility affects about 15-20% couples attempting pregnancy and in about half cases the problem lies in the male. Among the sperm parameters, linear progressive motility is one of the most important predictors of fertility potential. Though genetic and chromosomal abnormalities are important aetiological factors in the pathogenesis of male infertility, the mechanism involved in impaired sperm motility is poorly understood. Here we report mitochondrial DNA (mtDNA) mutations with increased seminal reactive oxygen species (ROS) levels and higher DNA fragmentation level in the sperm resulting in decreased ATP production which plays an important role in sperm motility defect. Thus it is important to understand the aetiology of asthenozoospermia and to distinguish if infertile men harbour nuclear or mtDNA mutation as they are very important prognostic markers. This case study also highlights that routine semen parameters are very modest predictors of fertility outcome but ROS estimation and DNA integrity analysis by Comet assay have better diagnostic and prognostic capabilities. Thus this study is a detailed and comprehensive workup of an infertile asthenozoospermic male.     

Influence of motility and vitality in intracytoplasmic sperm injection with ejaculated and testicular sperm

The vitality of spermatozoa used for intracytoplasmic sperm injection (ICSI) is a crucial factor for fertilization, establishment and outcome of a pregnancy in assisted reproductive technique cycles. The sperm origin may also be a limiting factor, although little is known about this issue. It is known that the motility of injected spermatozoa and their origin from ejaculate or testicular biopsies are important predictors in terms of fertilization, pregnancy and birth rates. Oocytes of patients in 2593 cycles were retrieved in our in vitro fertilization programme and inseminated via ICSI. We used motile (group 1, n = 2317) or immotile ejaculated spermatozoa (group 2, n = 79), motile sperm retrieved from testicular biopsies (group 3, n = 62) and immotile spermatozoa from testicular biopsies (group 4, n = 135). Female age and number of oocytes retrieved did not differ significantly among the groups. The fertilization rates were as follows: 67.1% in group 1, 49.8% in group 2, 68.3% in group 3 and 47.8% in group 4. The pregnancy rates in cases where three embryos had been transferred amounted to 35.7% in group 1, 17.3% in group 2, 38.3% in group 3 and 20.5% in group 4. The embryo quality showed no differences between groups 1 and 3 (14.5), and between groups 2 (11.8) and 4 (10.8). The abortion rate was similar in groups 1-3, but increased in group 4 (26.6%, 27.3%, 31.6% and 55.5%). Irrespective of their origin, the fertilization potential of injected spermatozoa was found to be influenced by motility. The resulting pregnancy and birth rates, i.e. the potential of the resulting embryos to implant and to achieve viable pregnancies, seem to be additionally dependent on the sperm origin. This was well shown by declining rates when spermatozoa in a relatively early stage of maturity had been used. We see increasing evidence that the degree of sperm maturity has an important impact on the outcome of ICSI. In obstructive azoospermia, spermatozoa retrieved from the epididymis should be used rather than testicular biopsy spermatozoa, or testicular sperm should be preincubated in culture medium before ICSI.     

Testicular versus ejaculated spermatozoa in ICSI cycles of normozoospermic men with high sperm DNA fragmentation and previous ART failures

As a part of male assessment, conventional sperm parameters including morphologic features have been dedicated as major factors influencing fertilisation and pregnancy rates in assisted reproductive technology (ART). Genomic integrity of spermatozoa has also been found to influence fertility prognosis, and hence, sperm DNA fragmentation index (DFI) has been adopted by many centres to document this entity. Despite several suggested approaches, there is lack of universal consensus on optimising fertility outcomes in males with high sperm DFI. In this context, the results from cycles using testicular spermatozoa (TESA) obtained by aspiration were compared with those of ejaculated spermatozoa (EJ) in normozoospermic subjects with high sperm DFI and previous ART failures. Clinical (41.9% versus 20%) and ongoing pregnancy rates (38.7% versus 15%) were significantly better and miscarriages were lower in TESA group when compared to EJ group. Sperm DFI should be a part of male partner's evaluation following unsuccessful ART attempts. When high DFI is detected (>30%), ICSI using testicular spermatozoa obtained by TESA seems an effective option particularly for those with repeated ART failures in terms of clinical, ongoing pregnancies and miscarriages even though conventional sperm parameters are within normal range.     

Comparison of pregnancy and neonatal outcomes of intracytoplasmic sperm injection performed with frozen versus fresh testicular sperm

BackgroundIt remains controversial whether there is a difference in the prognosis of intracytoplasmic sperm injection (ICSI) using frozen or fresh testicular sperm in patients with obstructive azoospermia (OA). Moreover, in the available studies, few have tracked neonatal outcomes. This study aimed to compare the pregnancy and neonatal outcomes of ICSI using cryopreserved sperm versus fresh sperm collected by testicular sperm aspiration (TESA).MethodsA total of 317 OA patients treated with ICSI in a university affiliated hospital from January 2016 to December 2020 were included in this study. The participants were divided into two groups according to the type of sperm used for ICSI: frozen sperm group (n=154) and fresh sperm group (n=163). The pregnancy and neonatal outcomes of the two groups were compared.ResultsThe data produced by this study showed no significant statistical difference in the 2 pronuclei (2PN) fertilization rate, 2PN cleavage rate, high-quality blastocyst rate, and the average number of transferred embryos in the frozen and fresh sperm groups. Similarly, no difference was found in implantation rate, clinical pregnancy rate, multiple pregnancy rate, miscarriage rate, premature delivery rate, live birth rate, and gender ratio at birth (P>0.05). The average newborn birth weight was similar in both groups (2,932.61±728.40 vs. 3,100.32±515.64 g, respectively) (P>0.05). A higher incidence of low birthweight (LBW) newborns was found in the frozen sperm group (20.91% vs. 8.49%) (P<0.05). Multiple logistic regression analysis showed that LBW is related to single or twin pregnancies (P<0.01), but not sperm (frozen or fresh) (P>0.05). We further analyzed the twin and single pregnancies in the two groups separately, and found that the incidences of LBW were both similar (P>0.05). There was no difference in the Apgar scores at 1 min and 5 min after birth between the two groups (P>0.05).ConclusionsThe use of frozen testicular sperm by TESA was efficient for men with OA. There were similar pregnancy and neonatal outcomes following TESA-ICSI using frozen or fresh sperm in this retrospective study. Prospective investigations are needed for further validation.     

An additional marker for sperm DNA quality evaluation in spermatozoa of male partners of couples undergoing assisted reproduction technique (IVF/ICSI): Protamine ratio

The aim of this study was to evaluate the relationship between the protamine ratio (P1/P2), DNA fragmentation of spermatozoa and protamine deficiency. Patients were grouped into fertile (G1; n = 151) and sub‐fertile (G2; n = 121). DNA fragmentation in spermatozoa was analysed by a TUNEL assay (terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labelling), and the protamination was determined by CMA3 staining, while Western blot was used to measure protamine P1 and P2. While sperm DNA fragmentation (SDF) and protamine ratio were significantly elevated in G2 compared with G1 (12.31 ± 7.01% vs. 17.5 ± 9.5%; p = .001) and (0.91 ± 0.43 vs. 0.75 ± 0.42; p = .003); respectively, the CMA3 positive showed no difference at all between G1 and G2. In G1, the CMA3 positive correlated negatively with the P1/P2 ratio and SDF (r = ?.586, r = ?.297; p = .001 respectively). In contrast, the protamine ratio correlated positively with SDF (r = .356; p = .001). In G2, no correlation was observed between CMA3 positive, SDF and the P1/P2 ratio but the P1/P2 ratio showed a positive correlation with SDF (r = .479; p = .001). In conclusion, the spermatozoa DNA deterioration was closely associated with abnormal protamination but showed an association with the protamine ratio, more than with CMA3 positive. Therefore, for the evaluation of DNA damage in spermatozoa, the P1/P2 ratio might act as an additional biomarker.     

Cryptorchidism: incidence and sperm quality in infertile men

In a population of 8500 men attending the andrology outpatient clinic, 200 men (2.35%) were recorded as having some disturbances with the descent of the testes into the scrotum. Medical history of the patients revealed that 51 underwent unilateral orchidopexy; 40 bilateral orchidopexy; and 24 were treated with human chorionic gonadotropin in order to induce descent of their testes. In addition, 6 patients reported spontaneous descent of the testes, and 13 others were found to be unilaterally cryptorchid upon physical examination. Results of semen analysis, hormonal profile, testes position, and testicular volume were compared to those of 105 proven fertile men. The major finding of this study shows that post-partum undescended testes suffer from primary Sertoli cell malfunction as reflected by elevated serum follicle stimulating hormone levels. Serum luteinizing hormone and testosterone levels were within the normal range. Surgical descent of the testes did not improve sperm production, proved by low sperm quality of all the study groups, compared to the cryptorchid group. Among the patients who were operated on, no correlation was found between age at operation and semen variables. All groups showed poor sperm quality which can be defined as oligoteratoasthenozoospermia. The degree of spermatogenic damage was in the following order of diagnosis or treatment: bilateral orchidopexy greater than cryptorchid testes greater than hormonal treatment greater than unilateral orchidopexy greater than late spontaneous descent of the testes. Thus, it is advisable to postpone surgical treatment of cryptorchidism and apply this only after a waiting period, and if the hormonal approach has failed to descend the testis.     

Stability of sperm characteristics in men with disturbances in sperm quality

Sixty-one men referred to our laboratory for semen analysis, and subsequently judged to exhibit some form of sperm pathology, were asked to return for a second analysis, not less than 2 months after the first, in order to assess the stability of the pathological changes observed. In almost half of the cases, the referring physician had, on his own initiative, started hormone or antibiotic treatment. The sperm parameters studied included sperm count, sperm motility judged by laser-Doppler spectroscopy, and sperm morphology and viability. The motility characteristics included percentage motile, their average velocity, and percentage swimming in a progressive manner, and their progressive velocity. In untreated subjects, there was no significant difference between the first and second analysis in any of the sperm parameters measured. This was also true for both oligozoospermic individuals (less than 20 x 10(6) sperm/ml) and the group with higher sperm concentrations. All parameters were highly correlated on the two occasions. The average coefficients of variation of the paired observations were highest for sperm count (approximately 25%) and lowest for sperm velocities and the proportion of abnormal and viable cells in the ejaculate (1-9%). No major differences in the extent of variation could be detected between the low and high sperm density groups. In general, the unsystematic antibiotic and hormone regimens (clomiphene or androgen) used by the referring physicians had no discernable effect on any aspect of sperm quality, indicating the need for more controlled and standardized programmes of treatment.     

Relationship between hyaluronic acid binding assay and outcome in ART: a pilot study

The sperm–hyaluronan binding assay (HBA) is a diagnostic kit for assessing sperm maturity, function and fertility. The aim of this prospective cohort pilot study was to evaluate the relationship between HBA and WHO sperm parameters (motility, concentration and detailed morphology) and possible influence of sperm processing on hyaluronic acid binding. A cohort of 68 patients undergoing a first combo in vitro fertilisation/intracytoplasmic sperm injection treatment after failure of three or more intrauterine insemination cycles were included in the study. Outcome measures studied were fertilisation rate, embryo quality, ongoing pregnancy rate and cumulative pregnancy rate. HBA outcome improved after sperm preparation and culture, but was not correlated to detailed sperm morphology, concentration or motility. HBA did not provide additional information for identifying patients with poor or absent fertilisation, although the latter had more immature sperm cells and cells with cytoplasmic retention present in their semen. HBA outcome in the neat sample was significantly correlated with embryo quality, with miscarriage rates and ongoing pregnancy rates in the fresh cycles, but not with the cumulative ongoing pregnancy rate. No threshold value for HBA and outcome in combo IVF/ICSI treatment could be established. The clinical value for HBA in addition to routine semen analysis for this patient population seems limited.     

Efficacy of varicocele embolization versus ligation of the left internal spermatic vein for improvement of sperm quality

Efficacy of surgical varicocelectomy versus embolization of the spermatic vein was studied in 137 men diagnosed as suffering from left varicocele. The men were divided randomly into three groups according to the methods of treatment: A--embolization of the internal spermatic vein (51 men); B--Ivanissevich technique of high ligation of the spermatic veins (43 men); and C--Bernardi technique of high ligation (43 men). The groups were similar in terms of age, duration of infertility and possessed semen characterized as oligoteratoasthenozoospermia. The fertility of the female partners was evaluated carefully and they were found to be potentially fertile. Varicocele was diagnosed by at least two of the following methods: physical palpation during valsalva manoeuvre, venography, or scrotal scanning using the technetium pertechnetate radioactive method. Semen quality was assessed before treatment and at 3, 6 and 9 months post-treatment. Fecundity was followed-up for 18 months. The major results were: (i) Shrinkage of the varicocele was found in all three groups studied. The same rate of recurrence was recorded in the three groups (24%, 37% and 35% in groups A, B and C, respectively). (ii) Improvement of sperm quality was significant in groups A and B, with better results in group B. (iii) The pregnancy rate was significantly higher in group B, compared with A (38.2% vs. 20.6%; P less than 0.05). Thus, high ligation of the internal spermatic vein yields better results than low ligation or embolization as far as semen quality and pregnancy is concerned.     

Relationship between donor sperm parameters and pregnancy outcome after intrauterine insemination: analysis of 2821 cycles in 1355 couples

The aim of this study was to investigate whether sperm parameters can affect the pregnancy outcome of artificial intrauterine insemination with cryopreserved donor spermatozoon (AID). A total of 1355 couples received 2821 AID treatment cycles in the Reproductive Medicine Center of the Tongji Medical College between January 2010 and December 2013, and the data were collected and retrospectively analysed. The relationship between pre‐freezing, post‐thawing as well as optimised sperm parameters and AID pregnancy outcome was investigated. Clinical pregnancy rate and cumulated pregnancy rate were also calculated. A total of 728 cycles from 2821 treatment cycles achieved pregnancies, and cumulated pregnancy rate was 25.81%. Pre‐freezing progressive sperm motility in pregnant cycles was higher than that in nonpregnant cycles (P = 0.001); logistic regression analysis also indicated that pre‐freezing progressive sperm motility was the only parameter affecting pregnancy outcome (P = 0.0001). Our study also showed that the cumulated pregnancy rate increased progressively and reached a plateau after the fifth cycle. In conclusion, pre‐freezing progressive sperm motility should be a valuable predictor for AID pregnancy outcome. Female fertility factors should be considered, or IVF/ICSI should be recommended when couples received more than 5 AID cycles without pregnancy.     

Sperm DNA fragmentation in subfertile men: the effect on the outcome of intracytoplasmic sperm injection and correlation with sperm variables

OBJECTIVE

To present the first UK data on sperm DNA fragmentation levels in subfertile men and fertile controls, the correlation with semen variables, and to assess the effect on the outcome of intracytoplasmic sperm injection (ICSI).

PATIENTS, SUBJECTS AND METHODS

In all, 56 subfertile men undergoing ICSI (28 with positive and 28 with a negative outcome for paternity) and 10 control fertile semen donors were recruited. The sperm DNA fragmentation index (DFI) was assessed on raw pre‐preparation samples using the sperm chromatin structure assay. A mean of 5212 sperm were analysed per sample and DFI data are presented by fertility status, ICSI outcome and correlated with semen variables (assessed using World Health Organisation criteria).

RESULTS

Total DFI was significantly higher in subfertile men than in fertile controls (mean and median of 22.8% and 17.0% vs 8.4% and 5.0%; P < 0.001), as was the proportion of both moderate DFI (16.4% and 13.0% vs 6.4% and 4.0%; P = 0.001) and high DFI (6.2% and 6.1 vs 2.0% and 1.0%; P = 0.01). This difference remained significant when the control men were compared only with the subfertile men with successful paternity. There was no significant difference in DFI in the subfertile men when analysed by ICSI outcome (mean and median of 24.5% and 17.0% vs 22.3% and 21.0% for successful and unsuccessful cycles, respectively; P = 0.94). There was a positive statistically significant correlation (r = 0.37; P = 0.02) between the DFI and sperm morphology.

CONCLUSIONS

This study confirms a relationship between male subfertility and sperm DFI; we discuss the correct role for genetic testing of sperm in the evaluation of subfertile men. Although DNA fragmentation data might help to decide a suitable treatment, once it is decided to proceed with ICSI, DFI levels have no effect on the outcome.     

ICSI周期显微取精术治疗13例AZF微缺失致无精子症的临床研究

目的探讨ICSI周期显微镜下睾丸切开取精术在AZF微缺失致无精子症患者中的临床应用价值。方法对2014年1月至2016年4月在我院生殖医学中心男科就诊并接受ICSI周期显微镜下睾丸切开取精术的13例AZF微缺失致无精子症患者的临床资料进行回顾性研究,分析指标主要包括AZF微缺失类型、显微镜下睾丸切开取精术次数、单/双侧睾丸取精、精子获得率、精子可用率、正常受精率、可用胚胎率、优胚率及临床妊娠率。结果 ICSI周期显微镜下睾丸切开取精术的13例AZF微缺失致无精子症患者中,AZF微缺失类型分别为:AZFa区完全缺失1例,AZFc区完全缺失9例,AZFc区部分缺失1例,AZFd区部分缺失1例,AZFc区完全缺失伴AZFd区部分缺失1例。共行ICSI周期显微镜下睾丸切开取精术14例次,其中9例次单侧睾丸取精术,5例次双侧睾丸取精术;精子获得率78.57%,可用率72.73%。行ICSI助孕治疗者的正常受精率61.29%、可用胚胎率71.43%、优胚率40.48%、临床妊娠率57.14%。结论 ICSI周期显微镜下睾丸切开取精术可以使部分AZF微缺失致无精子症患者获得数量足够的精子行ICSI助孕治疗,并能获得良好的正常受精率、可用胚胎率、优胚率及临床妊娠率,是一种有效的治疗方法。     

Chromosomal abnormalities in couples undergoing intracytoplasmic sperm injection. A study of 370 couples and review of the literature

Intracytoplasmic sperm injection (ICSI) is now widely acknowledged as the most effective therapeutic approach to severe male infertility or unsuccessful in vitro fertilization. Cytogenetic investigations were performed in 370 females and 335 males prior to ICSI between January 1997 and April 2003. Nine men (2.7%) and 48 women (13%) had an abnormal karyotype, 44 females having some degree of numerical sex chromosome mosaicism. A review of the literature showed the prevalence of all types of chromosomal abnormalities to be much higher among male and female partners of couples examined prior to ICSI than among newborns. As most ICSIs are performed with ejaculated spermatozoa from oligospermic men, the distribution and the prevalence of the several types of chromosomal abnormalities are closer to those of oligospermic rather than azoospermic males. Our results combined with those of the literature stress the importance of karyotyping both male and female partners before ICSI is started. Adequate genetic counselling, possibly followed by prenatal diagnosis, should be offered if a chromosomal anomaly is detected.     

精液分析中精子浓度室内质量控制方法的研究

目的:建立一种可行的精子浓度检测的室内质控方法。方法:设定20×106和80×106两种浓度的冷冻精液作为高、低值室内质控品(IQC),QC-BEADSTM质控珠(以下简称质控珠)作为对照。采用双盲法,4名技术员分别通过计算机辅助精液分析(CASA)对上述假定的室内质控品及质控珠进行精子浓度的测定,绘制出各自的质控图(Xbar图和Sbar图),连续检测1个月,计算出相应的高、低浓度值批内及批间变异系数(CV%),并进行比较。结果:①假定室内质控品高、低值批内变异系数分别CV3.5%,CV2.4%;批间分别为CV10.2%、CV9.6%。质控珠高、低值批内分别为CV5.1%,CV7.1%,批间分别CV7.1%,CV8.2%。两组高、低值的批内变异均<10%,批间变异均<15%,符合(Levey-Jennings)L-J质量控制原则,均能达到室内质控目的。②两组间高、低值的批内变异系数及批间变异系数无显著性差异(P>0.05),提示假定质控品可代替质控珠行实验室内部质量控制。结论:我们建立的精子浓度测定的室内质控方法是简单、易行、可靠。