Scientific molecular basis for treatment of reproductive failure in the human: an insight into the future.

PURPOSE: The purpose of this review is to summarize science-based new treatments for human reproductive failure and future developments. RESULTS: First will be discussed popular but erroneous myths of current non-science based treatments. Then will be discussed new treatments and their scientific base, including ovary and egg freezing, and transplantation to preserve fertility in young women undergoing gonadotoxic chemotherapy and radiation for cancer; new perspectives on human epididymal sperm maturation based on a comparison between ICSI (intracytoplasmic sperm injection) with testis sperm versus epididymal sperm; simplifying IVF and reducing cost by more intelligent and milder ovarian stimulation; improving pregnancy rate in older women; searching the genome to find genes which control spermatogenesis and whose deletion or mutation causes spermatogenic failure; and human spermatogenic stem cell culture to treat azoospermia, and to preserve fertility in pre-pubertal boys undergoing cancer treatment. CONCLUSION: With stem cell biology and molecular understanding of reproductive failure, new therapies for previously untreatable infertility are currently on the near horizon. Conversely our clinical results with new therapeutic approaches are adding to our understanding of the basic science of reproduction. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.

Ovary cryopreservation and transplantation for fertility preservation.

The aim of this review is to summarize the state-of-the-art of ovarian transplantation and cryopreservation. This field has progressed over the last half century from simple animal experiments to sophisticated application in humans. The initial poor results in humans began to improve when a series of nine monozygotic (MZ) twin pairs discordant for premature ovarian failure (POF) underwent ovary transplantation at one center. All of these fresh ovary transplants were successful, resulting in 11 healthy babies in 7 of the 9 recipients. The same surgical techniques were then applied to 3 frozen ovary tissue transplants, up to 14 years after the ovary had been frozen, resulting in 3 more healthy babies. Around the world, the number of healthy babies has now risen to 28. Even ovary allotransplantation is being attempted in the not so uncommon situation where a previous bone marrow donor is now willing to donate ovarian tissue to the same recipient. Recipients routinely reinitiated ovulatory menstrual cycles and normal Day 3 serum FSH levels by 4.5 months. Most conceived naturally (three of them twice or three times from the same graft). The duration of function of fresh ovarian grafts, contrary to initial expectations, indicated minimal oocyte loss from ischemia time. Grafts of just modest portions of ovarian tissue have lasted >7 years. In vitro studies suggest that vitrification of ovarian tissue may be an improvement over the 70% oocyte viability loss from slow freeze.

A series of monozygotic twins discordant for ovarian failure: ovary transplantation (cortical versus microvascular) and cryopreservation.

BACKGROUND: A series of monozygotic (MZ) twin pairs discordant for premature ovarian failure presented an unusual opportunity to study ovarian transplantation. METHODS: Ten MZ twin pairs requested ovarian transplantation and eight have undergone transplantation with cryopreservation of spare tissue. Seven had a fresh cortical tissue transplant, one of whom received a second frozen-thawed transplant after the first ceased functioning at three years. One had a fresh microvascular transplant. RESULTS: All recipients reinitiated ovulatory menstrual cycles and normal Day 3 serum FSH levels by 77-142 days. Six have already conceived naturally (one twice). Currently, two healthy babies have been delivered, and another three pregnancies are ongoing. The oldest transplant functioned for 36 months, resulting in one child and one miscarriage. She conceived again after a frozen-thawed secondary transplant. There was no apparent difference in return of ovarian function between the eight fresh ovarian grafts and the one frozen graft. CONCLUSIONS: Ovarian transplantation appears to restore ovulatory function robustly. Successful pregnancies, including one after cryopreservation, bode well for application to fertility preservation.

Graft-versus-host reactivity and renal allograft survival in rats given allogeneic spleen cells or spleen allografts.

Selective recruitment of antigen-sensitive cells (ASC) into the spleen as a method of inducing specific suppression was attempted by intravenous injection of either DA or Lewis spleen cells 24 hr before a (DA X Lewis)F1 renal allograft into a Lewis or DA recipient, either with or without a splenectomy. This led to suppression of rejection in the DA recipient and delayed rejection in the Lewis recipient. Splenectomy produced a minimal augmentation effect. Assay of graft-versus-host (GVH) reactions in (DA X Lewis)F1 rats by a popliteal node assay showed that injection of allogeneic DA or Lewis spleen cells 48 hr before the assay significantly reduced the reaction produced by node lymphocytes but not spleen lymphocytes, suggesting a loss of ASC from the lymph nodes. Lewis spleen allografts did not produce such a significant reduction in the GVH reactivity of DA node lymphocytes as intravenous Lewis cells, whereas DA spleen allografts led to an increased GVH reactivity of Lewis node lymphocytes. From these studies, it is not possible to attribute the suppression produced by the intravenous injection of allogeneic cells to selective recruitment of antigen-sensitive cells to the spleen.

Microsurgical reversal of tubal sterilization: factors affecting pregnancy rate, with long-term follow-up.

Among 48 women with long-term follow-up after microsurgical reversal of tubal sterilization, the pregnancy rate correlated closely to the length of tube on the longest side. Aside from an accurate anastomosis, no other factor significantly affected pregnancy rate.

Growth of baby kidneys transplanted into adults.

To determine whether the growth of a kidney is affected by the age of the host or is independently programmed, baby rat kidneys were isologously transplanted into adult rats that underwent unilateral or bilateral nephrectomies. The growth of these transplanted baby kidneys was compared to the growth of baby kidneys that were left intact in the growing baby rat. After allowing for compensatory hypertrophy, the kidneys, whether in babies or adults, were found to grow at the same rate and to the same ultimate size. Renal size could be predictably related to renal age, but not to the age of the host.

Microsurgery for vasectomy reversal and vasoepididymostomy.

Accurate microsurgical techniques developed by us have allowed a considerable improvement in success rate for vasectomy reversal, and a better understanding of the pathophysiology of obstruction. This has permitted us also to treat pathologic obstructive azoospermia more effectively.

Microsurgery in clinical urology.

Microsurgery is not a new tool to the urologist. Transurethral resection is probably the most challenging type of microsurgery, and we do it daily. The manipulations of microvascular dissections and anastomoses may be more intricate, but the basic concept of performing delicate procedures while looking down a tube is "old hat" to us. The new techniques discussed here may extend the range of operative solutions to difficult clinical problems in urology perhaps as transurethral resection did in the thirties and forties.

Role of epididymis in sperm maturation.

One hundred ninety patients with obstructive azoospermia caused by bilateral epididymal blockage have been followed up for four years or longer after undergoing "specific tubule" vasoepididymostomy. When anastomosis was required in the corpus epididymis, the "patency" rate was 78 percent, and the overall pregnancy rate was 56 percent. The pregnancy rate for "patent" cases was 72 percent, indicating that a high fertility rate can be obtained with sperm that have not transited the full length of corpus epididymis. By contrast, with vasoepididymostomy to the caput epididymis there was a 73 percent "patency" rate, but the overall pregnancy rate was only 31 percent. The pregnancy rate for "patent" cases was 43 percent. Sperm from the corpus epididymis have a higher rate of fertility than sperm from the caput epididymis, but sperm from proximal areas of the corpus have no less fertility than sperm from the distal corpus epididymis. The most remarkable observation is that in almost half the cases sperm that have never journeyed beyond the caput epididymis seem to be capable of causing pregnancy.

Epididymal extravasation following vasectomy as a cause for failure of vasectomy reversal.

Twenty-eight men undergoing vasectomy reversal who were found to have no sperm in the proximal vas fluid on one or both sides underwent microscopic epididymal exploration. In 33 of 39 cases so explored, normal sperm were found in the epididymal fluid of the corpus, despite absence of sperm in the vas fluid. Epididymal histology distal to this site revealed extensive interstitial sperm granulomas resulting from rupture of the epididymal duct. Testicular biopsy revealed normal spermatogenesis. Secondary epididymal obstructions were noted when there was copious fluid in the vas deferens proximal to the vasectomy site as well as when there was scanty fluid. It is concluded that persistent azoospermia after an accurate microscopic vasovasostomy results from the secondary epididymal obstruction induced by rupture of the epididymal duct related to the pressure increase after vasectomy.

PIP: 28 men underwent microscopic epididymal exploration and serial transections with biopsy. All of these men were noted to have no sperm in the vas fluid on the testicular side of the vasectomy site at the time of intended vasovasostomy. The age range of the patients was 27-55 years. All patients underwent several semen analyses preoperatively to determine that they were azoospermic. In 11 of these 28 patients the need for epididymal exploration was bilateral; in 17 the exploration was only unilateral. Thus, a total of 39 epididymides were examined. In 33 of the 39 cases, despite absence of sperm in the vas fluid, an abundance of sperm was noted in the epididymal fluid at some point between the junction of the tail of the epididymis with the corpus epididymidis and the area of the proximal corpus epididymidis. Epididymal histology distal to this site showed extensive interstitial sperm granulomas resulting from rupture of the epididymal duct. Testicular biopsy revealed normal spermatogenesis. Secondary epididymal obstructions were noted when there was copious fluid in the vas deferens proximal to the vasectomy site as well as when there was scant fluid. Persistent azoospermia after an accurate microscopic vasovasostomy results from secondary epididymal obstruction induced by the rupture of the epididymal duct related to the pressure increase after vasectomy.

Vasoepididymostomy to the head of the epididymis: recovery of normal spermatozoal motility.

We previously reported a microsurgical technique for bypassing epididymal obstruction by performing a specific microanastomosis to the epididymal tubule. When obstruction was near the head of the epididymis, spermatozoal motility was always poor (0 to 1%) even though the numerical count was high. There are now over 1 1/2 years of follow-up on the first five patients who had a vasoepididymostomy performed in the proximal region (head) of the epididymis. None of these patients had more than 0 to 1% spermatozoal motility postoperatively despite counts of more than 50 million sperm/ml. However, within 1 1/2 to 2 years the spermatozoa of these patients eventually developed normal motility. This study verifies that in humans spermatozoa derived from the head of the epididymis are at first not capable of motility. However, after 1 or 2 years, these spermatozoa eventually recover normal motility. This unexpected finding sheds new light on epididymal physiology and offers some hope for men with proximal epididymal obstruction.

Transplantation of a human testis for anorchia.

One of two genetically identical twins (30 years old) had been born with two normal testes and the other with none. In the anorchic twin, preoperative serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were extremely high, and the serum testosterone level was extremely low. In the twin with two testes, preoperative serum FSH, LH, and testosterone levels were normal. After successful transplantation of a testis from the twin with two testes to the twin with no testes, using the microvascular technique, the recipient twin developed a normal serum testosterone level within 2 hours of surgery; his FSH and LH levels came down toward a normal range more slowly over the ensuing 4 weeks. The donor's FSH level became mildly elevated 2 days postoperatively but returned to normal by 3 months. Thereafter, serum FSH, LH, and testosterone levels remained persistently normal in both twins. In the donor, pre- and postoperative sperm counts were normal. Preoperatively the recipient's semen had no sperm, but postoperatively the sperm content has slowly increased to normal levels.

Microscopic vasoepididymostomy: specific microanastomosis to the epididymal tubule.

A new microscopic technique for vasoepididymostomy is described. Conventional approaches to epididymal obstruction rely on the formation of a fistulous tract after the walls of the vas deferens are grossly sutured to the outer tunic of the epididymis; such methods have a low success rate. With a direct and accurate end-to-end anastomosis of the inner lumen of the vas deferens specifically to the epididymal tubule there is a greater likelihood of normal patency. Furthermore, such an anastomosis can be performed at the lowest possible level of the epididymis, allowing a greater opportunity for sperm maturation.

PIP: A new, direct anastomosis of the epididymal tubule to the mucosa of the vas deferens is described as performed on 14 men with epididymal obstruction. In this method, about 1 cm of the epididymis is dissected and cut transversely, as low as possible above the obstruction, if it can be visualized nder the x10 or x16 operating microscope. This cut will expose 3-10 epididymal tubules. The one bearing the sperm fluid can be ascertained by examining smears. If no sperm are present, another section .5 cm higher is examined until sperm gush out, although it is important to cut as low as possible in the epididymis to insure motile sperm. The vas is mobilized enough to reach the epididymis without tension. Dissection of both vessels can be done with x2.5 ocular loupes. Anastomosis, under operating microscope, must be performed from the posterior aspect first, using 9-0 or 10-0 nylon, then carefully suturing from outside to inside the epididymal tubule and from inside to outside the vas mucosa, so that the two lumens approximate perfectly. The outer muscularis of the vas is then sutured to the epididymal tunic with 10 to 12 9-0 nylon-interrupted sutures. The man may be discharged in 1-2 days, advised to rest for 1 week. Monthly sperm counts need be repeated until normal motile sperm appear. In this preliminary series of 14 men, 11 had normal motile sperm counts. 1 patient did not have motile sperm: his anastomosis was in the head of the epididymis. The 2 with low counts had not shown a reliable flow of sperm fluid from the anastomosed tubule.

Perfect anatomical reconstruction of vas deferens with a new microscopic surgical technique.

Early results with a new, two-layer microscopic technique for anastomosis of the vas deferens, using an operating microscope and ultrafine suture, reveal that patency of the vas deferens can be achieved in virtually every case. Normal sperm counts can be achieved in up to 95% of patients. Failure with conventional techniques is usually due to continuing partial obstruction. A poor sperm count after this technique is most likely due to an inherent inability of the patient to produce normal sperm, a likely sequela of chronic obstruction. Thus, while success is also good 10 years after vasectomy, it is not as predictable. Previous failure with a conventional operation does not limit success with a reoperation using the microscopic two-layer tehcnique. Operations on 200 patients since this original study are confirming these early results.

Microscopic vasectomy reversal.

More than 300 patients undergoing microscopic vasovasostomy have been carefully studied in an effort to determine the factors which affect the recovery of fertility after an accurate microscopic reanastomosis. The over-all pregnancy rate in an unselected group of early patients was 71%. Recovery of fertility correlated with the return of normal sperm counts and with the quality of seminal fluid in the vas deferens on the testicular side of the obstruction at the time of vasovasostomy. The three most important factors influencing return of fertility after vasovasostomy are (1) a meticulous microscopic technique for reconnection, (2) the duration of time the vas deferens has been obstructed, and (3) the presence of absence of a sperm granuloma at the site of the vasectomy, venting the long-term pressure buildup which otherwise would occur. The presence of a sperm granuloma at the vasectomy site generally ensured the presence of good quality sperm in the vas fluid at the time of vasovasostomy and the recovery of a good sperm count postoperatively. If all three of these factors are favorable, vasectomy should be reversible for most patients.

Simultaneous treatment of the wife in infertile couples with oligospermia.

Poor ovulation was found to be common in infertile couples with oligospermia. When the sperm count was less than 5 x 10(6)/ml, 33% of wives had poor ovulation. When the sperm count was between 5 x 10(6)/ml and 20 x 10(6)/ml, 78% of wives had poor ovulation. Improvement in sperm count was most likely to increase the chances of conception when the pretreatment sperm count was less than 5 x 10(6)/ml. Pregnancy in infertile couples with oligospermia cannot be related simply to treatment or improvement in the husband's oligospermia but can only be viewed in the context of the wife's fertility, which is quite frequently also impaired in infertile couples with male factor problems.

Microsurgical reversal of female sterilization: the role of tubal length.

A group of 25 women who had undergone tubal sterilization by a variety of techniques underwent microsurgical reanastomosis. The length of tube remaining and the segments of tube involved were carefully noted preoperatively, but were not used as a basis for selection of patients. The only criterion utilized for selecting patients was the presence of fimbriae on at least one side. In all patients, anatomical patency was achieved at surgery. Normal intrauterine pregnancy was directly related to tubal length. Of seven patients who had less than 3 cm of tube, none achieved pregnancy. Of seven patients who had 3 to 4 cm of tube, three achieved a normal intrauterine pregnancy. Among 11 patients who had over 4 cm of tube, all 11 achieved a normal intrauterine pregnancy. No significant difference in pregnancy rate was noted in women who had short segmants of ampulla so long as total tubal length was adequate.

PIP: 25 women who had undergone tubal sterilization by a variety of techniques underwent microsurgical reanastomosis. It was the intention of this study to determine whether tubal length had any effect on the outlook for a successful pregnancy. The length of tube remaining and the segments of tube involved were carefully noted preoperatively, but were not used as a basis for selection of patients. The only criterion utilized for selecting patients was the presence of fimbriae on at least 1 side. Anatomical patency was achieved at surgery in all patients. 15 of the 25 patients (60%) achieved uterine pregnancy, with only 1 experiencing an ectopic pregnancy. Normal intrauterine pregnancy was directly related to tubal length. Of 7 patients who had less than 3 cm of tube, none achieved pregnancy. Of 7 patients who had 3-4 cm of tube, 3 achieved a normal intrauterine pregnancy. Among 11 patients who had more than 4 cm of tube, all achieved a normal intrauterine pregnancy. No significant difference in pregnancy rate was noted in women who had short segments of ampulla so long as total tubal length was adequate. It seems that as long as there are healthy fimbriae, microsurgical reanastomosis can restore tubal patency in all cases.

Open-ended vasectomy, sperm granuloma, and postvasectomy orchialgia.

The presence of a sperm granuloma at the vasectomy site prevents epididymal pressure build-up, perforation, and the formation of an epididymal sperm granuloma. It thus enhances reversibility of the vasectomy and lessens the likelihood of epididymal discomfort. In two prospective vasectomy series, a sperm granuloma was intentionally allowed to form by not sealing the testicular end of the vas. The sperm granuloma resulted in no instance of orchialgia, but created a greater risk of spontaneous recanalization. This latter problem could only be solved by more careful sealing of the upper end of the vas. In a separate series of nine patients vasectomized elsewhere and specifically referred to us for chronic and persistent postvasectomy orchialgia, seven had no sperm granuloma at the vasectomy site. Pain in these cases was localized in the epididymis and was relieved by vasovasotomy. Any technique of vasectomy carries a very small risk of orchialgia, whether due to the presence of a sperm granuloma at the vasectomy site or to increased epididymal pressure.

PIP: In an Ottawa study, 410 patients consented to open-ended vasectomy, and in a St. Louis study, 23 patients underwent open-ended vasectomy, in which the abdominal end is cauterized but the lumen on the testicular side is not ligated, clipped, or cauterized. In the Ottawa series, 3% of the patients developed no sperm granuloma and 97% did develop sperm granuloma. The Concept unit was used on 148 patients with a 4% failure rate; however, the Hemoclip application was used on 262 patients with only a 0.4% failure rate. In the St. Louis series, all 23 patients developed sperm granulomas with l case of recanalization. In 9 patients referred to St. Louis for chronic and persistent postvasectomy orchialgia with pain localized in the epididymis, the pain was relieved by vasovasostomy. Evidence indicated that heat cautery was not as efficient a method of sealing the vas as the Hemoclips due to the high failure rate.

Ultrastructure of human sperm in men with congenital absence of the vas deferens: clinical implications.

OBJECTIVE: To determine the ultrastructural features of epididymal and vasa efferentia sperm in men with congenital absence of the vas deferens. DESIGN: Prospective. SETTING: University of California Irvine Center for Reproductive Health. PATIENTS: Thirteen men with surgical diagnosis of congenital absence of the vas deferens undergoing epididymal and vasa efferentia aspiration for assisted reproductive technology procedures. RESULTS: The morphological quality and the integrity of the spermatozoa aspirated from the extratesticular segment of the rete testis, the vasa efferentia, and the caput epididymis were always markedly superior to those of sperm aspirated from the corpus and cauda epididymis, where the vast majority, if not all, were degenerating or frankly necrotic. The aspirates obtained from the distal segments of the epididymis also contained large numbers of sperm-laden macrophages; these were instead absent or exceptional in the fluids aspirated from the pre-epididymal portions of the excurrent pathways and from the caput of the epididymis. CONCLUSIONS: This study demonstrates that the ultrastructural morphology of spermatozoa obtained by aspiration from the rete testis, vasa efferentia, and caput epididymis of individuals with congenital absence of the vas deferens is indistinguishable from that of spermatozoa in the semen.