Pregnancy with frozen-thawed and fresh testicular biopsy after motile and immotile sperm microinjection, using the mechanical touch technique to assess viability

Sir, We read with interest the article titled ‘Pregnancy withfrozen-thawed and fresh testicular biopsy after motile and immotilesperm microinjection, using the mechanical touch technique     

ICSI outcome in patients with transient azoospermia with initially motile or immotile sperm in the ejaculate

BACKGROUND: In patients with transient azoospermia, few sperm may be found in the ejaculate. We investigated the outcome of ICSI in patients with transient azoospermia. METHODS: Records of patients with transient azoospermia referred during a 42 month period were reviewed. If only immotile sperm were found, the sample was incubated with 30% human serum albumin (HSA) before motility re-assessment. If still immotile, mechanical assessment of sperm viability was utilized. Study groups were: (A) motile sperm; (B) motility achieved by HSA; (C) no motility, but viability assessed by a mechanical technique; and (D) control group with sperm counts from 1 to 5 x 10(6)/ml. There were 57 couples (cycles) in the study group and 43 couples (cycles) in the control group. RESULTS: Age, days of stimulation and endometrial thickness were comparable among groups. In 29.8% of the cycles, only immotile sperm were found. Fertilization and cleavage rates were higher in groups A and D than in groups B and C. Clinical pregnancy rate/cycle and live birth rate/cycle were not different among groups. No congenital malformations were found in newborns. CONCLUSION: Fertilization and cleavage rates were lower in patients with initially immotile sperm compared with those with initially motile sperm and oligoasthenoteratozoospermia patients. Clinical pregnancy and viable pregnancy rates were not statistically different among groups, although when only immotile sperm were present both clinical pregnancy and live birth rate were lower in comparison with cycles with motile sperm.     

Pregnancy outcome and neonatal data of children born after ICSI using testicular sperm in obstructive and non-obstructive azoospermia

BACKGROUND: Registries on outcome of ICSI pregnancies obtained with testicular sperm do not differentiate between obstructive (OA) and non-obstructive azoospermia (NOA). We evaluated the pregnancy outcome and neonatal data on children born after ICSI using testicular sperm of men with histologically proven OA or NOA. METHODS: Pregnancies obtained after ICSI using testicular sperm of men with defined NOA (n = 70) were compared with those of men with OA (n = 204). RESULTS: Multiple birth rates in NOA and OA couples, respectively, were 21 versus 27% (P = NS), overall preterm delivery rates were 38 versus 26% (NS), and prematurity rates were 24 versus 13% for singletons (NS) and 86 versus 54% for twins (relative risk 1.59, 95% confidence interval 1.04-2.42). Median gestational age for singletons was 38.3 versus 39.3 weeks, respectively (P < 0.05). The low birth weight rates were 34 versus 31%, respectively (NS). The early perinatal mortality rate was 66 versus 15 per 1000 births, respectively, (NS). Major congenital malformations were observed in 4 versus 3%, respectively, of the live born babies (NS). Prenatal karyotypes showed 7% de-novo abnormalities in the NOA group versus 1% in the OA group (NS). CONCLUSIONS: Our data do not show differences between NOA and OA pregnancies except for a strong tendency towards a lower gestational age in singletons and a higher percentage of premature twins in the NOA group. Although our data are based on a limited sample, the differences observed call for further analysis. Given the low pregnancy rates after ICSI with NOA, a multicentre study, differentiating NOA and OA patients, would be recommended.     

Andrology: CASE REPORT Pregnancy after intracytoplasmic sperm injection of spermatozoa extracted from frozen-thawed testicular biopsy

The case report illustrates the successful application of anew method of sperm extraction from a frozen-thawed testicularbiopsy specimen within an established programme of intracytoplasmicsperm injection.     

Predictive value of testicular histology in secretory azoospermic subgroups and clinical outcome after microinjection of fresh and frozen-thawed sperm and spermatids

BACKGROUND: A retrospective study was carried out on 159 treatment cycles in 148 secretory azoospermic patients to determine whether histopathological secretory azoospermic subgroups were predictive for gamete retrieval, and to evaluate outcome of microinjection using fresh or frozen-thawed testicular sperm and spermatids. METHODS: Sperm and spermatids were recovered by open testicular biopsy and microinjected into oocytes. Fertilization and pregnancy rates were assessed. RESULTS: In hypoplasia, 97.7% of the 44 patients had late spermatids/sperm recovered. In maturation-arrest (MA; 47 patients), 31.9% had complete MA, and 68.1% incomplete MA due to a focus of early (36.2%) or late (31.9%) spermiogenesis. Gamete retrieval was achieved in 53.3, 41.2 and 93.3% of the cases respectively. In Sertoli cell-only syndrome (SCOS; 57 patients), 61.4% were complete SCOS, whereas incomplete SCOS cases showed one focus of MA (5.3%), or of early (29.8%) and late (3.5%) spermiogenesis. Only 29.8% of the patients had a successful gamete retrieval, 2.9% in complete and 77.3% in incomplete SCOS cases. In total, there were 87 ICSI, 39 elongated spermatid injection (ELSI) and 33 round spermatid injection (ROSI) treatment cycles, with mean values of fertilization rate of 71.4, 53.6 and 17%, and clinical pregnancy rates of 31.7, 26.3 and 0% respectively. CONCLUSIONS: Histopathological subgroups were positively correlated with successful gamete retrieval. No major outcome differences were observed between testicular sperm and elongated spermatids, either fresh or frozen-thawed. However, injection of intact round-spermatids showed very low rates of fertilization and no pregnancies.     

Effects of vasectomy on spermatogenesis and fertility outcome after testicular sperm extraction combined with ICSI

BACKGROUND: Each year 40,000 men have a vasectomy in the UK whilst another 2400 request a reversal to begin a second family. Sperm can now be obtained by testicular biopsy and subsequently used in assisted conception with ICSI. The study aims were to compare sperm yields of men post-vasectomy or with obstructive azoospermia (OA) of unknown aetiology with yields of fertile men and to assess any alteration in the clinical pregnancy rates after ICSI. METHODS: Testicular tissue was obtained by Trucut needle from men who had undergone a vasectomy >5 years previously or had OA from other causes and from fertile men during vasectomy. Seminiferous tubules were milked to measure sperm yields. Numbers of Sertoli cells and spermatids and thickness of the seminiferous tubule walls were assessed using quantitative computerized analysis. RESULTS and CONCLUSIONS: Sperm yields/g testis were significantly decreased in men post-vasectomy and in men with OA, relative to fertile men. Significant reductions were also observed in early (40%) and mature (29%) spermatid numbers and an increase of 31% was seen in the seminiferous tubule wall (basal membrane and collagen thickness) of vasectomized men compared with fertile men. Clinical pregnancy rates in couples who had had a vasectomy were also significantly reduced.     

Pregnancies achieved after frozen-thawed pronuclear oocytes obtained by intracytoplasmic sperm injection with spermatozoa extracted from frozen-thawed testicular tissues from non-obstructive azoospermic men.

The use of frozen-thawed testicular tissue as a source of spermatozoa for intracytoplasmic sperm injection (ICSI) in non-obstructive azoospermia yields favourable fertilization and pregnancy rates while avoiding both repetitive biopsies and unexpected cycle cancellations. Spermatozoa were obtained from frozen-thawed testicular biopsy specimens from 67 non-obstructive azoospermic men. Following fertilization, supernumerary two pronuclear (2PN) oocytes were frozen. After thawing, 17 cycles of embryo transfer were carried out with a mean number of 2.7 embryos and a mean cumulative embryo score (CES) of 18.3 per transfer. The clinical pregnancy and implantation rates per transfer in these cycles (23.5 and 8.3% respectively) were comparable to those of fresh embryo transfers (35.7 and 12.7% respectively) with a mean number of 2.7 embryos and a mean CES of 28.7 per transfer. Abortion rates, although higher with cryopreserved 2PN oocytes were not significantly different. With this approach, cryopreservation of supernumerary 2PN oocytes can be used to improve the cumulative pregnancy rates in a severely defective spermatogenetic population. To our knowledge, these are the first pregnancies reported which have been obtained by the transfer of cryopreserved pronuclear oocytes obtained from ICSI using cryopreserved testicular spermatozoa.     

A detailed cytogenetic analysis of large numbers of fresh and frozen-thawed human sperm after ICSI into mouse oocytes

BACKGROUND: Since information about chromosome aberrations in micro-manipulated sperm is still inadequate, cytogenetic analysis was performed on large numbers of fresh and frozen-thawed (FT) human sperm after injection into mouse oocytes. The effects of the ICSI procedure on oocytes are also discussed based on analysis of the mouse chromosome complements. METHODS: After the injection of fresh and FT human sperm into mouse oocytes, chromosomes of the hybrid oocytes were analysed at first cleavage metaphase. RESULTS: Incidences of the hybrid oocytes at the first cleavage metaphase were significantly different between fresh (71.5%) and FT sperm groups (80.1%) (P < 0.05). The chromosome analysis of 477 fresh and 141 FT sperm showed no difference in the incidences of aneuploidy (1.6/0.7%), structural aberrations (8.8/7.8%) or diploidy (0.0/0.0%) between these categories. The cytogenetic result did not differ from our previous result using IVF between human sperm and hamster oocytes. In an additional cytogenetic study on 615 mouse chromosome complements, the incidence of diploidy (5.4%) was significantly higher than those (0.3-2.8%) in the previous mouse cytogenetic studies, and the hybrid oocytes with no mouse chromosomes (2.0%) existed. CONCLUSIONS: This result suggests that the ICSI procedure induces no sperm chromosome aberrations but increases numerical aberrations in oocyte chromosome complements.     

Pregnancies achieved with testicular and ejaculated spermatozoa in combination with intracytoplasmic sperm injection in men with totally or initially immotile spermatozoa in the ejaculate

The efficacy of intracytoplasmic sperm injection (ICSI) employingtesticular and ejaculated spermatozoa was assessed in 24 coupleswith totally or initially immotile spermatozoa. No criteriawere employed in selecting which patients would be treated withtesticular or ejaculated spermatozoa. The men were chosen atrandom. Testicular spermatozoa obtained by testicular spermextraction were used in 14 and ejaculated spermatozoa were usedin 10 of these couples. In all cases, asthenozoospermia wastotal in their basal semen sample. In 12 male partners, spermatozoawere totally immotile before and after Percoll gradient fractionation(totally immotile). In the remaining 12 men, spermatozoa initiallyshowed a total absence of motility; however, some of the spermatozoahad showed very poor motility (0.1%) after Percoll gradientfractionation and a 13–2.0 h incubation period (initiallyimmotile). Of these 24 total asthenozoospermic males, 14 alsohad total terato-zoospermia. The fertilization and cleavagerates in the testicular and ejaculated sperm groups were 533and 963 and 543 and 94.4% respectively. One cycle resulted incomplete fertilization failure, and in 23 embryo transfer cyclesa total of 10 pregnancies were obtained (41.6%). Eight pregnancieswere achieved in the testicular sperm group, while only twopregnancies were obtained in the ejaculated sperm group. Fourpregnancies, two from the ejaculated sperm group and two fromthe testicular sperm group, resulted in clinical abortions inthe first trimester. Of the remaining six pregnancies, two havealready resulted in healthy births and four pregnancies arenow in the second or third trimester in the testicular spermgroup. Using testicular spermatozoa in combination with ICSIcan be an alternative mode of treatment in cases with totallyor initially immotile spermatozoa in the ejaculate. Very lowpregnancy rates have been obtained and no ongoing pregnancyhas been achieved using ejaculated spermatozoa in these cases.     

Pregnancies after ICSI using sperm with abnormal head-tail junction from two brothers: case report

We report ICSI pregnancies in two couples with a history of long standing primary infertility in which the sperm of the male partner were either acephalic or had abnormal head-midpiece attachments. The two couples, in which the men are brothers, underwent ICSI. Sperm were analysed by transmission electron microscopy and immunocytochemistry with an anti-MPM2 monoclonal antibody. The first couple underwent two ICSI cycles, each consisting of the injection of two mature oocytes and the transfer of two embryos. A successful pregnancy occurred after the second transfer and led to the birth to a healthy girl. The second couple underwent three ICSI cycles, each consisting of the injection of 18 oocytes and the transfer of two embryos; the last of these led to a triple ongoing pregnancy which included two identical twins. Caesarean section led to the birth of three fetal-growth restricted children. This case report demonstrates that ongoing pregnancies can be achieved in cases of abnormal development of the head-neck attachment. The genetic origin of this syndrome is generally accepted, but the phenotypic heterogeneity observed by light and electron microscopy among published cases suggests that there are a variety of genetic causes of this syndrome.     

High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy

In cases requiring microsurgical epididymal sperm aspiration(MESA) for congenital absence of the vas deferens (CAVD) orirreparable obstructive azoospermia, often no spermatozoa canbe retrieved from the epididymis, or there may even be no epididymispresent. We wished to see whether testicular biopsy with testicularsperm extraction (TESE) in such cases could yield spermatozoathat would result in successful fertilization and pregnancy(despite the absence of epididymal spermatozoa) using intracytoplasmicsperm injection (ICSI). In the same setting during the same2-week period, 28 patients with CAVD or irreparable obstructionwere treated; 16 consecutive fresh MESA—ICSI cycles and12 cycles which required testicular biopsy with testicular spermextraction (TESE—ICSI) were performed. Normal two-pronuclearfertilization rates were similar in both groups: 45% for epididymalspermatozoa and 46% for testicular biopsy-extracted spermatozoa.Cleavage rates were also similar (68% for epididymal and 65%for testicular spermatozoa). The ongoing pregnancy rates inthis series were 50 and 43% respectively. We conclude that epididymalspermatozoa and testicular spermatozoa yield similar fertilization,cleavage and ongoing pregnancy rates using ICSI. When epididymalspermatozoa cannot be retrieved, a testicular biopsy can beperformed and the few barely motile spermatozoa thus obtainedcan be used for ICSI. It appears that all cases of obstructiveazoospermia can now be successfully treated.     

Pregnancy and birth after intracytoplasmic sperm injection with spermatozoa from a patient with tail stump syndrome

Tail stump syndrome, which may be associated with primary ciliarydyskinesia, is also associated with morphological defects ofthe flagellum resulting in severe asthenozoospermia. Until recently,these morphological anomalies caused definite male infertility.Today, however, new methods such as micromanipulation techniquesprovide a rational therapy for this patient group. A pregnancyfollowed by living offspring was achieved following the intracytoplasmicinjection of immotile spermatozoa from a patient with tail stumpsyndrome.     

The relative viability of human spermatozoa from the vas deferens, epididymis and testis before and after cryopreservation

Testicular and epididymal spermatozoa are routinely used with in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to achieve pregnancies. In addition, excess cryopreserved spermatozoa can be thawed and used for ICSI. However, information on the recovery of epididymal and testicular spermatozoa after freeze-thaw is lacking. This is important to determine the feasibility of using previously cryopreserved aspirated spermatozoa for ICSI. We prospectively compared the viability of fresh and frozen-thawed spermatozoa from the vas deferens, epididymis and testicle by several measures. Testis spermatozoa were obtained from men with non-obstructive azoospermia (n = 5), epididymal spermatozoa from men with obstructive azoospermia (n = 8), and vasal spermatozoa from fertile men by vasal irrigation at vasectomy (n = 5). The viability of fresh spermatozoa was assessed by motility, two vital stains (carboxyfluorescein, 0.08 mg/ml and propidium iodide, 20 mg/ml) and the hypo-osmotic swelling assay (HOS; 100 mmol/l citrate and fructose). After cryopreservation, spermatozoa were thawed and all viability measures repeated. Although fresh vasal spermatozoa were the most motile, testicular spermatozoa exhibited similar, high viability (91 and 86% respectively) by vital stain. Spermatozoa from testis, epididymis and vas deferens survived cryopreservation equally well by vital stain, but not by motility. As a selection measure, the HOS assay identified significantly more viable epididymal and testicular spermatozoa than did motility in both fresh and frozen-thawed populations. It appears feasible to use frozen-thawed extracted spermatozoa for ICSI when motility and a selection measure such as the HOS assay are used. With fresh testis spermatozoa, selection methods may not be necessary prior to ICSI, as cell viability is high.     

Cumulative probability of achieving an ongoing pregnancy after in-vitro fertilization and intracytoplasmic sperm injection according to a woman's age, subfertility diagnosis and primary or secondary subfertility

The aim of this study was to estimate reliable cumulative probabilities of achieving an ongoing pregnancy after successive in-vitro fertilization or intracytoplasmic sperm injection (IVF/ICSI) cycles, according to a woman's age, subfertility diagnosis and primary or secondary subfertility. Therefore reasons for quitting treatment without achieving an ongoing pregnancy were taken into account. Moreover, we studied whether there were trends in cumulative probabilities after adjustment for potential confounding effects of the other two characteristics, duration of subfertility, year of first treatment and reason for quitting treatment. In total, 2984 IVF/ICSI cycles were performed in 1315 couples at the University Hospital Nijmegen, The Netherlands, between 1991 and 1998. The 'realistic' cumulative probability of achieving an ongoing pregnancy was 54.5% after five consecutive IVF/ICSI cycles, which was about 10% lower (absolute value) than the optimistic probability calculated by life-table analysis and about 10% higher (absolute value) than the most pessimistic estimate. Women of 35 years or younger had a higher probability of achieving an ongoing pregnancy than the older women. As ICSI is now an option, there were no obvious differences between the subfertility diagnosis subgroups. The cumulative probability after the first two IVF/ICSI cycles was higher in women with secondary subfertility than in those with primary subfertility; this advantage disappeared after further treatment. These trends remained valid after adjustment for confounding factors.     

Obstetric outcome of pregnancies after the transfer of cryopreserved and fresh embryos obtained by conventional in-vitro fertilization and intracytoplasmic sperm injection.

This study reports the obstetric outcome of pregnancies obtained after the transfer of cryopreserved or fresh embryos where the initial procedure was standard in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Pregnancies obtained after frozen IVF (n = 245) or frozen ICSI (n = 177) were compared with a control group of pregnancies after fresh embryo transfer in standard IVF (n = 245) and ICSI (n = 177) cycles were selected as controls. The controls were matched according to maternal age, parity and date of embryo transfer. In the standard IVF group, the biochemical pregnancy rates in the cryopreserved and fresh groups were 18.8 and 9.8% respectively (P < 0.01). In the ICSI group, the biochemical pregnancy rates in the cryopreserved and fresh groups were 16.4 and 6.8% respectively (P < 0.01). The miscarriage rates were comparable between the cryopreserved and fresh groups. However, in the frozen ICSI group the miscarriage rate (26.0%) was significantly higher than in the frozen conventional IVF group (13.1%) (P = 0.001). The frequencies of preterm deliveries, infants with very low birthweight and intrauterine deaths were similar in the groups. The low birthweight rates in the frozen IVF (16.1%) and ICSI (12.1%) groups were significantly lower than those in the fresh IVF (32.2%) and ICSI (32.7%) groups (P < 0.001). The major malformation rates in the frozen IVF (2.4%) and ICSI (2.9%) groups were not different from the major malformation rates in the fresh IVF (4.5%) and ICSI (2.4%) groups. In conclusion, the cryopreservation process had no negative impact on the outcome of pregnancies over 20 weeks of gestation. Long-term follow-up studies are needed in order to prove the safety of the freezing-thawing process.     

Men with infertility caused by AZFc deletion can produce sons by intracytoplasmic sperm injection, but are likely to transmit the deletion and infertility.

Deletion of the AZFc region of the Y chromosome is the most frequent molecularly defined cause of spermatogenic failure. We report three unrelated men in whom azoospermia or severe oligozoospermia was caused by de-novo AZFc deletions, and who produced sons by intracytoplasmic sperm injection (ICSI). We employed polymerase chain reaction (PCR) assays to examine the Y chromosomes of their four infant sons. All four sons were found to have inherited the Y chromosome deletions. Such sons are likely to be infertile as adults. This likelihood should be taken into account when counselling couples considering ICSI to circumvent infertility due to severe oligozoospermia or non-obstructive azoospermia.     

Lack of an association between environmental exposure to polychlorinated biphenyls and p,p'-DDE and DNA damage in human sperm measured using the neutral comet assay

BACKGROUND: Chlorinated organic chemicals, such as polychlorinated biphenyls (PCB), hexachlorobenzene (HCB), dichlorodiphenyl trichloroethane (DDT), and dichlorodiphenyl dichloroethene (DDE, the most stable daughter compound of DDT) are persistent lipophilic compounds found in a large portion of the general population. To explore the hypothesis that environmental exposure to these compounds is associated with altered DNA integrity in human sperm, a study of 212 male partners of a sub-fertile couple who presented to the Massachusetts General Hospital Andrology Laboratory was conducted. METHODS: The neutral single cell microgel electrophoresis assay (comet assay) was used to assess DNA integrity in sperm. VisComet image analysis software was used to measure total comet length, the proportion of DNA present in the comet tail, and tail distributed moment, an integrated measure of length and intensity. RESULTS: In the regression analyses, there were no statistically significant consistent associations between the comet assay parameters and any of the individual PCB congeners, sum of PCB, or p,p'-DDE. CONCLUSION: These results suggest that there are not strong relationships between adult levels of these chlorinated organic compounds and sperm DNA damage as measured by the comet assay.     

Study of the vasculature of the caprine reproductive organs using the tissue‐clearing technique,with special reference to the angioarchitecture of the utero‐ovarian vessels and the adaptation of the ovarian and/or vaginal arteries to multiple pregnancies

Arteries of the reproductive tracts of nonpregnant does and does at 4, 7, 10, 13, 16, and 18 weeks of gestation were injected in situ with Microfil®. The tracts were fixed, dehydrated, and rendered transparent to reveal the paths of arteries. The tortuous ovarian artery lay in close apposition to the uterine tributary of the ovarian vein, an arrangement that may serve as a local utero‐ovarian pathway for the corpus luteum (CL) luteolysis at the end of nonfertile estrous cycle. During pregnancy, this arteriovenous arrangement might transfer luteotropic substances from uterus to ovary, which might serve in maternal recognition of pregnancy and fit the fact that the goat is CL‐dependent throughout gestation. In some cases of triplets, the size of the uterine branch of the ovarian artery was equal to or even larger than that of its parent artery and/or the ipsilateral uterine artery, and the vaginal artery contributed a connecting branch to the uterine artery. These physiological adaptations of the ovarian and/or vaginal arteries, which have not previously been described, correlate well with the increasing nutrient demands of the growing multiple fetuses. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.