Ovarian tissue cryopreservation: a practical option?

Strategies to preserve fertility in young women undergoing potentially curative chemotherapy for malignant disease have been extremely limited. This limitation stems from the complex physiology of the human oocyte and the difficulties encountered in attempting to cryopreserve both developing and mature oocytes in sufficient quantities. Although in vitro fertilization and embryo cryopreservation can be used in those young women with a partner, this technique is unsuitable for the vast majority of patients and offers only a small chance of a pregnancy. Advances in cryobiology coupled with encouraging results in laboratory animals have prompted research into the storage of ovarian cortical tissue, which in young women is rich in primordial follicles. This tissue can be grafted back into the host, theoretically restoring the possibility of normal fertility. Primordial follicles contain oocytes at their least differentiated stage and appear to be relatively resistant to the combined insults of cryopreservation and the subsequent grafting procedure. Interest in this technique has been fuelled by its successful application in large domestic animals, such that ovarian tissue banking is being rapidly adopted into clinical practice before there is any hard evidence of its efficacy in humans.     

Ovarian tissue cryopreservation: a practical option?

Strategies to preserve fertility in young women undergoing potentially curative chemotherapy for malignant disease have been extremely limited. This limitation stems from the complex physiology of the human oocyte and the difficulties encountered in attempting to cryopreserve both developing and mature oocytes in sufficient quantities. Although in vitro fertilization and embryo cryopreservation can be used in those young women with a partner, this technique is unsuitable for the vast majority of patients and offers only a small chance of a pregnancy. Advances in cryobiology coupled with encouraging results in laboratory animals have prompted research into the storage of ovarian cortical tissue, which in young women is rich in primordial follicles. This tissue can be grafted back into the host, theoretically restoring the possibility of normal fertility. Primordial follicles contain oocytes at their least differentiated stage and appear to be relatively resistant to the combined insults of cryopreservation and the subsequent grafting procedure. Interest in this technique has been fuelled by its successful application in large domestic animals, such that ovarian tissue banking is being rapidly adopted into clinical practice before there is any hard evidence of its efficacy in humans.     

Optimizing potential for fertility: fertility considerations for the pediatrician

Whether for the prepubertal or pubertal child, the goal of fertility preservation is to obtain cells or tissues to be used to produce future children. For the prepubertal child, preservation efforts involve germ cells, earlier forms of sperm, and immature follicles, rather than mature spermatozoa or follicles. Options for prepubertal children include for boys freezing testicular tissue and extracting testicular sperm or for girls obtaining ovarian cortical or follicular tissue for storage. These procedures involve extraction and storage of immature gametes for subsequent in vitro maturation, although attempts for sperm currently involve only animal studies. For adolescent subjects who have sufficient gonadal development and reserve, sperm, oocytes, and ovarian cortex can be retrieved as among adults.     

Cryopreservation of semen from adolescent patients with malignancies

In adult oncological patients semen cryopreservation offers the possibility of preserving fertility prior to aggressive therapy that may lead to infertility. The cryopreserved semen can later be used to induce pregnancies in the partner by techniques of assisted fertilization. In adolescent boys the question of fertility is often beyond consideration when the young patient's life is threatened acutely. However, improved survival rates increasingly prompt the question of quality of life after therapy, including fertility. Semen quality is known to be impaired in patients with malignancies and may be further impaired by the process of cryopreservation. Since normal values for semen in adolescents are not known and spermatogenesis may be impaired by the malignant disease, it was unclear whether semen samples from adolescents with malignancies warrant cryopreservation at all. In order to demonstrate the feasibility of semen cryopreservation in adolescent males, we compared the results from 12 pubertal boys aged 14–17 years with those from 17 young adults aged 18–20 years who had similar malignancies and, additionally, to 210 adults with malignancies (>20 years). Luteinizing hormone serum values were significantly lower in adolescents than in adult patients. Follicle stimulating hormone showed a significant increase with age. Testosterone serum levels and testicular volumes showed similar distribution patterns in adolescent and adult men. Sperm concentrations, sperm motility, and normal sperm morphology in the adolescent patients did not show significant differences compared with adults. Thus cryopreservation of semen should be considered as an option to young male patients whose cancer therapy will include potentially gonadotoxic treatment. © 1996 Wiley-Liss, Inc.     

Sperm banking in adolescent cancer patients.

BACKGROUND: Semen cryopreservation is a widely available method of maintaining fertility in male cancer patients. However this facility is not always used. AIMS: To identify the barriers to successful sperm banking in a group of adolescent and young adult patients. METHODS: Questionnaires were administered to 55 patients aged 13-21 years who had received potentially gonadotoxic therapy between 1997 and 2001 and had been offered sperm banking. RESULTS: Forty five questionnaires were completed; 67% of respondents were able to bank sperm. Those who had been unsuccessful were younger and described higher levels of anxiety at diagnosis and greater difficulty in talking about fertility. They also described less understanding of sperm banking at the time of diagnosis. CONCLUSION: Most adolescent cancer patients who have been offered fertility preservation are able to bank sperm. Younger patients may be helped by the provision of high quality information and more open discussion of the technique.     

Preserving female fertility following cancer treatment: Current options and future possibilities

Children and women of reproductive age are increasingly surviving cancer diagnoses, and therefore long‐term quality‐of‐life issues are of greater importance at the time of diagnosis. Cancer therapies including radiation and chemotherapy can be detrimental to fertility, and therefore many patients are motivated to preserve fertility prior to cancer treatment. The only highly successful method in preserving fertility to date is embryo cryopreservation, which may not be appropriate for some patients due to age, delay in treatment, cancer type and stage, as well as availability of an acceptable sperm donor. Alternative methods including oocyte cryopreservation and ovarian tissue banking may also preserve fertility while providing additional flexibility to patients. In vitro ovarian follicle maturation following tissue banking is one potential approach that would not require a delay in cancer therapy for ovarian stimulation, would not require an immediate sperm donor, and does not carry the risk of reintroducing malignant cells following tissue transplantation. In vitro follicle culture systems have resulted in successful live births in the mouse. However, many challenges must be addressed in translating the system to the human. This review summarizes current approaches to fertility preservation and discusses recent developments and future challenges in developing a human in vitro follicle culture system. Pediatr Blood Cancer 2009;53:289–295. © 2009 Wiley‐Liss, Inc.     

Ovarian Tumorlike Structures, Biovular Follicles, and Binucleated Oocytes in Children

The occurrence of microscopic gonadoblastomalike and sex cord tumor with annular tubuleslike structures, biovular follicles, and binucleated oocytes was analyzed in 288 ovaries from 222 consecutive pediatric autopsies. Tumorlike structures were identified in 29% of the autopsies studied, biovular follicles were observed in 52%, and binucleated oocytes in 19%. These changes were not observed in 22 ovaries from adults. Sixteen cases with gonadoblastomalike structures were stained immunohistochemically for placental alkaline phosphatase and were nonreactive for this marker. These tumorlike structures and multiovular follicles are probably an expression of abnormal folliculogenesis that apparently disappears as the child matures and should not be confused with true neoplasms. Although these morphologic structures resemble the patterns of specific types of gonadal neoplasms, there is little evidence to suggest any potential for future neoplastic transformation.     

Ovarian tumorlike structures, biovular follicles, and binucleated oocytes in children: their frequency and possible pathologic significance

The occurrence of microscopic gonadoblastomalike and sex cord tumor with annular tubuleslike structures, biovular follicles, and binucleated oocytes was analyzed in 288 ovaries from 222 consecutive pediatric autopsies. Tumorlike structures were identified in 29% of the autopsies studied, biovular follicles were observed in 52%, and binucleated oocytes in 19%. These changes were not observed in 22 ovaries from adults. Sixteen cases with gonadoblastomalike structures were stained immunohistochemically for placental alkaline phosphatase and were nonreactive for this marker. These tumorlike structures and multiovular follicles are probably an expression of abnormal folliculogenesis that apparently disappears as the child matures and should not be confused with true neoplasms. Although these morphologic structures resemble the patterns of specific types of gonadal neoplasms, there is little evidence to suggest any potential for future neoplastic transformation.     

Sperm banking in adolescent cancer patients

Background

Semen cryopreservation is a widely available method of maintaining fertility in male cancer patients. However this facility is not always used.

Aims

To identify the barriers to successful sperm banking in a group of adolescent and young adult patients.

Methods

Questionnaires were administered to 55 patients aged 13–21 years who had received potentially gonadotoxic therapy between 1997 and 2001 and had been offered sperm banking.

Results

Forty five questionnaires were completed; 67% of respondents were able to bank sperm. Those who had been unsuccessful were younger and described higher levels of anxiety at diagnosis and greater difficulty in talking about fertility. They also described less understanding of sperm banking at the time of diagnosis.

Conclusion

Most adolescent cancer patients who have been offered fertility preservation are able to bank sperm. Younger patients may be helped by the provision of high quality information and more open discussion of the technique.     

Cryopreservation of semen from pubertal boys with cancer

BACKGROUND: The possibility of cryopreservation of semen from adolescents has until now received only little attention. Therefore, we have investigated the possibility of cryopreservation of semen in adolescent boys with cancer. PROCEDURE: Forty-five boys, aged 13-18 years, admitted because of cancer during the period January 1, 1995 to July 31, 1998 were eligible. Semen was obtained after masturbation in the majority of the cases. In three boys, semen was preserved after penile vibration or electroejaculation in general anaesthesia. The semen samples were analysed for concentration, motility, and morphology according to the WHO guidelines. The sample was transferred into straws prior to cryopreservation at 196 degrees C in liquid nitrogen. RESULTS: Twenty-one boys delivered a semen sample for cryopreservation. Four boys were offered and accepted sperm banking but were not able to produce a sample. In 20 cases time did not allow an attempt of sperm banking, the boy was not assessed to be mature enough to deliver a semen sample, or the procedure was not accepted. The boys delivered 1-3 samples, and the total number of spermatozoa ranged from 0-210 millions. Median percentage of motile sperm was 50% (range 9-86%). Semen quality improved with age; however, a 13- year- old boy produced 75 million spermatozoa with 38% motile cells. CONCLUSIONS: Pubertal maturation should be assessed in all boys admitted for cancer, and the possibility of sperm banking should be discussed with the patient and his parents.     

Normal US appearance of ovaries and uterus in four patients with Turner's syndrome and 45,X karyotype

Pelvic ultrasonographic (US) studies of four patients (ages 11–19 years) with Turner's syndrome, 45,X karyotype, and normal ovarian function were reviewed. All four had persistent menses, spontaneous breast development, and normal follicular stimulant hormone (FSH) serum concentrations. The US studies depicted normal postpubertal uterus and normal-sized ovaries with follicles. In three patients, ovaries were seen bilaterally, while in one only one gonad was identified. Radiologists should be aware that patients with Turner's syndrome, even with a single X chromosome, may occasionally have normal genital development.     

Sexual differentiation of rat hepatic bile salt sulfotransferase isoenzymes

The mature female rat has three times the hepatic bile salt sulfotransferase (BSS) activity compared with male rats. This study examined the changes in two hepatic BSS isoenzyme activities during sexual maturation, and the role of estrogen in development of sex differences in BSS activities in mature rats. DEAE-Sephadex A-50 chromatography of hepatic cytosol from prepubescent pups revealed that more than 90% of total BSS activity was due to BSS I activity relative to BSS II, similar to postpubertal females. Sex differences in total BSS activities and the isoenzyme patterns developed after the onset of puberty at 30-35 days of age. BSS I was still the predominant isoenzyme in the adolescent female, similar to the prepubescent pup and mature female. In contrast, BSS I activity declined in adolescent males, which appeared to explain the fall in total BSS activity to only one-third of that of the female by maturity. BSS II activity was similar in both sexes at any age. Estrogen treatment of postpubertal male rats rapidly increased hepatic BSS capacity by enhancing BSS I activity producing an isoenzyme pattern similar to the mature female. This rapid enhancement of BSS I by estrogen was blocked by actinomycin D and puromycin. We concluded that 1) sex differences in BSS activities that develop during adolescence were in part due estrogen-maintaining BSS I activity in females and 2) estrogen regulates the synthesis of BSS I at a translational (or pretranslational) level.     

Testicular stem cells for fertility preservation: Preclinical studies on male germ cell transplantation and testicular grafting

Spermatogonial stem cells open novel strategies for preservation of testicular tissue and fertility preservation in boys and men exposed to gonadotoxic therapies. This review provides an update on the physiology of spermatogonial stem cells in rodent and primate testes. Species‐specific differences must be considered when new technologies on testicular stem cells are considered. Germ cell transplantation is presented as one novel and promising strategy. Whereas this technique has become an important research tool in rodents, a clinical application must still be regarded as experimental and many aspects of the procedure need to be optimized prior to a safe and efficient clinical application in men. Testicular grafting opens another exciting strategy for fertility preservation. Autologous and xenologous transfer of immature tissue revealed a high regenerative potential of immature testicular tissue. Grafting was applied in rodents and primates and resulted in the generation of sperm. Further research is needed before an application in humans can be considered safe and efficient. Despite the current limitations in regard to the generation of sperm from cryopreserved male germline cells and tissues, protocols for cryopreservation of testicular tissue are available and reveal a promising outcome. Since future improvements of germ cell transplantation and grafting approaches can be assumed, bioptic retrieval and cryopreservation of testicular tissue fragments should be performed in oncological patients at high risk of fertility loss since this is their only option to maintain their fertility potential. Pediatr Blood Cancer 2009;53:274–280. © 2009 Wiley‐Liss, Inc.     

Testicular histology related to fertility outcome and postpubertal hormone status in cryptorchidism

BACKGROUND: Early surgical correction of an undescended testis is performed to prevent the development of male infertility. However, in boys with cryptorchidism early successful surgery cannot prevent infertility if they lack Ad spermatogonia. In this study, sperm concentrations and postpubertal hormone levels were correlated to bilateral testicular histology. The aim was to define the risk of future infertility via a testis biopsy program for boys with cryptorchidism. METHODS: Eighty-nine boys who had an orchidopexy were subjected to bilateral testicular biopsy. Histological analysis of 178 biopsies indicated three groups of high, intermediate, and low risk of infertility according to the presence of Ad spermatogonia. After puberty, sperm concentrations were analysed and correlated with plasma gonadotropin and testosterone levels. FINDINGS: In patients with unilateral cryptorchidism 70% of scrotal testes had an impaired transformation of Ad spermatogonia, indicating that cryptorchidism is a bilateral disease. Sperm concentrations correlated to the number of Ad spermatogonia found at the time of orchidopexy (p<0.001). All males in the high risk of infertility group were oligospermic (mean: 8.9x10 (6) sperm/ejaculate) and 20% were azoospermic. These patients had 25 times less sperm compared to the group with presence of Ad spermatogonia in both testes (p<0.001). Correlations between testicular histology and postpubertal hormone levels confirmed a relative gonadotropin deficiency in the majority of males with cryptorchidism. INTERPRETATIONS: Ad spermatogonia proved to be a discriminating factor for the fertility outcome in cryptorchidism. Gonadotropin treatment following orchidopexy should be considered in cryptorchidism when no Ad spermatogonia are found in undescended gonads and scrotal testis have Ad germ cell counts <0.005 per tubule.     

Children conceived after intracytoplasmic sperm injection (ICSI): is there a role for the paediatrician?

AIM: The aim of the study was to evaluate current medical knowledge about children born after intracytoplasmic sperm injection (ICSI) with respect to congenital malformations, chromosome abnormalities and postnatal growth. RESULTS: The total malformation rate in children conceived after ICSI was comparable to the background population in nine of the 11 articles studied. In two of the studies, a significant increase in congenital malformations was found. More specifically, children born after ICSI appear to have a higher risk of urogenital malformations, especially hypospadias, which may be related to paternal subfertility. There is insufficient knowledge about chromosomal or genetic anomalies and auxological data in children born after ICSI. The methodological approach to follow-up the children was inconsistent, as the clinical examinations were not done with standardized ascertainment. CONCLUSION: Paediatricians and obstetricians should collaborate with fertility clinics to obtain valid longitudinal observations with respect to congenital malformations, neurological development, growth, pubertal maturation, fertility and morbidity in children conceived by ICSI.     

Evaluation of frozen thawed cauda epididymal sperms and in vitro fertilizing potential of bovine sperm collected from the cauda epididymal

In the present study, the fertilizing potential of semen recovered from slaughtered bulls epididymis was evaluated after cryopreservation, by conventional techniques and flow cytometry methods. The cauda epididymal was dissected and sperm were recovered and evaluated for volume, sperm concentration, and membrane and acrosome integrity using a flow cytometer. Sperm fertility potential was tested by in vitro fertilization (IVF). For each bull, three trials of IVF were performed. Before freezing, on average, the sperm concentration was 216 ± 27.5 × 10⁶ sperm/ml. Sperm viability averaged 86.5 ± 4%. The mean percentage of sperm with intact plasma membrane and acrosome before and after cryopreservation was 90.7 ± 2.9% and 90.8 ± 1.9% (P≥0.05), respectively. The fertilization rate using frozen/thawed epididymal semen averaged 64.1 ± 3.9% fertilization with no significant differences between bulls (P>0.05). For the bull considered as control, the fertilization rate was 72.2 ± 4.5%, differing significantly (P>0.05) from the frozen/thawed epididymal semen’s fertilization rate. In conclusion, it is possible to use in vitro techniques with cryopreserved spermatozoa obtained from bull’s epididymis using a controlled rate freezing method with a predetermined freezing curve, and with assessment of sperm’s viability by conventional techniques and flow cytometry methods, together with the fertilizing ability of cryopreserved epididymal spermatozoa.Key Words: Bovine, Cryopreservation, Epididymis, IVF, Semen     

Attitudes and practices of oncologists toward fertility preservation

Cancer is a life-threatening diagnosis. Fortunately, life-saving treatments are available to increase the chance of survival in many patients. Yet, many of these treatments are damaging to the reproductive organs and the patients' fertility. A cross-sectional study addressing the knowledge and practices of oncologists toward fertility preservation for male and female patients with cancer was conducted in Saudi Arabia. In 3 different regions of the country, oncologists were invited to participate in the study, through a self-administered questionnaire which was handed to them inquiring about their knowledge, attitude, and referral practices for sperm cryopreservation. Only one-half knew about intracytoplasmic sperm injection, oncologists rated their perception of the importance of cryopreservation as 7.8 ± 1.8. Their referral practice was very poor; less than 20% refer their patients to a specialist. Factors that were considered important to start discussion of cryopreservation were type of cancer, age of patient, number of children, marital status, and cost. Religion was not deemed as important as was anticipated. With regards to female fertility preservation, oncologists showed a positive attitude as revealed from their positive perception, however, their referral practices was very poor. Several gaps were present in the knowledge of oncologists, which could influence their attitude and in turn was reflected on their poor practice. Future training session should be organized to the oncologists for increasing their knowledge and enhancing their attitude.     

改良快速冷冻载体冻融人类稀少精子的实验研究

目的探讨人类稀少精子冷冻改良快速冷冻载体的实验研究,为重度少弱精子症患者的精子冷冻保存提供参考。方法2018年12月至2019年8月,在联勤保障部队第九〇〇医院生殖中心就诊的男性不育症患者:根据精液质量分为正常精液组和少弱精液组,每个实验样本选取3份精液分别进行充分混合,将混合精液稀释至浓度为(1~2)×10~6/ml,实验共计选取81份正常精液标本混匀稀释形成27个正常精液样本,选取54份少弱精液标本混匀稀释形成18个少弱精子样本。根据不同的冷冻载体各分3组进行精子冷冻:薄片精子冷冻管组(A组),麦管微量体积冷冻组(B组),改良快速冷冻载体组(C组),均采用商品化精子冷冻试剂1∶1混匀,熏蒸法后快速冷冻。比较3种载体冷冻复苏后,精子的活动力、存活率、DNA碎片指数和正常形态变化、精子顶体酶活性等指标变化,以评估改良快速冷冻载体的冷冻效率及安全性。统计学方法采用t检验、ANOVA分析、LSD多重比较。结果正常精液组的3种冷冻方法都会造成精子质量下降。正常精液组的C组的复苏后精子活动力、存活率均高于A组和B组,差异有统计学意义(P<0.05)。正常精液组:C组复苏精子DNA碎片指数略低于A和B组,但差异无统计学意义(P>0.05);A、B、C 3组复苏后精子正常形态率均低于冷冻前,差异有统计学意义(P<0.05)。少弱精液组:A、B两组复苏后精子DNA碎片指数均高于冷冻前,差异有统计学意义(P<0.01);C组精子DNA碎片指数高于冷冻前,但差异无统计学意义(P=0.068),C组复苏精子DNA碎片指数略低于A和B组,但差异无统计学意义(P>0.05),A、B、C 3组冷冻精子复苏后正常形态率均低于冷冻前,差异无统计学意义(P>0.05)。正常精液组:冷冻前精子顶体酶活性为(123.6±12.8)IU/10~6个精子,复苏后A、B、C 3组顶体酶活性均有下降,分别为(74.7±15.6)、(84.7±13.5)、(91.2±16.2)IU/10~6个精子,差异有统计学意义(F=37.896,P<0.001),从对于精子顶体影响看,B、C组优于A组,差异有统计学意义(P=0.043、P=0.001)。结论改良快速冷冻载体对于稀少精子冷冻保存有一定优势,值得进一步探讨研究。     

The effects of dietary vitamin B12 deficiency on sperm maturation in developing and growing male rats

ABSTRACT  To evaluate the role of vitamin B₁₂ on spermatogenesis, the effects of dietary vitamin B₁₂ deficiency on sperm maturation in developing rat fetuses and young growing rats were examined. The vitamin B₁₂-defi-cient diet was given to all the animals for three different periods: whole period (gestation to mature), gestation period (gestation to weaning), or immature period (3–12 weeks postnatal). Sperm examination revealed that the sperm count was markedly lower in male progeny (F1) that were vitamin B₁₂-deficient during the whole period. In addition, a significantly higher number of abnormal sperm, such as tailless and amorphous sperm, was observed. In male rats that were vitamin B₁₂-deficient during the immature period, the incidence of abnormal sperms was 14.4% and 4.8% for tailless and short tail, respectively. The motion rates, such as path velocity and straight line velocity, were decreased to 20–40% of the control value in rats that were vitamin B₁₂-deficient both during the whole and gestation periods. However, no effects of vitamin B₁₂ deficiency on sperm motility were observed during the immature and mature periods. From these findings, we suggest that dietary vitamin B₁₂ deficiency during pregnancy may induce irreversible damage in the germ cells of embryos and affect the maturation of spermatozoa.     

The effect of benzo(a)pyrene on fertility, primordial oocyte number, and ovarian response to pregnant mare's serum gonadotropin

The polycyclic aromatic hydrocarbon (PAH) benzo(a)pyrene (BP) reduced the fertility of DBA/2N mice in a dose-dependent fashion. Control mice produced offspring at a rate of 0.91 pups/mouse per week. Treatment with BP at doses of 10, 100, 200, and 500 mg/kg decreased offspring production rates to 0.61, 0.20, zero and zero pups/mouse per week, respectively. BP also destroyed primordial oocytes in similarly treated mice. Treatment with BP at doses of 10, 50, 100, and 500 mg/kg destroyed 20%, 58%, 88%, and 100%, respectively, of the primordial oocytes in DBA/2N mouse ovaries. Dose-response curves for fertility reduction and primordial oocyte destruction were identical. The threshold for fertility reduction was 3.4 mg/kg and for primordial oocyte destruction it was 2.7 mg/kg. The 50% effect doses for fertility reduction and primordial oocyte destruction were 25.5 mg/kg and 24.5 mg/kg, respectively. Although BP reduced fertility and destroyed primordial oocytes, it had no effect on the ovarian weight response to pregnant mare's serum gonadotropin (PMSG), consistent with the observations that BP at these doses does not destroy growing or preovulatory oocytes or follicles. The similarity of the dose-response curves for fertility reduction and primordial oocyte destruction suggested that their mechanism of action is similar and resides within the ovary. As BP had no effect on ovarian response to PMSG, the effect was likely to reside outside the regulatory mechanisms controlling ovulation. These data suggest that the site of fertility reduction by BP may reside in the mechanisms of fertilization, implantation, or early conceptus development.