Ovarian Damage and Decreased Activity

Gonadotoxicity – a decrease in ovarian activity – depends on several factors, including the age of the patient, the initial status of the ovaries (referred to as the antral follicular account), the treatment applied (chemotherapy, radiotherapy, or surgery) and cumulative doses, and the type of agent used.

As many factors may contribute, it is difficult to establish the exact incidence of premature ovarian failure after systemic chemotherapy. Most ovarian failure data are referenced to cancer patients, who receive higher doses of chemotherapy and, consequently, have an increased incidence of ovarian failure.

People with autoimmune disorders may also be treated with chemotherapy but usually at lower doses than cancer patients. So, chemotherapy’s consequences may not be so dreadful but determined by the cumulative dose. Although many of the patients treated with chemotherapy recover their ovarian function once chemotherapy is completed, there is an increased risk of premature ovarian failure, especially when related to age [11] and the use of alkylating agents [2, 12].

Age

The ovarian cortex has a limited number of primordial follicles that decrease with age. Previous chemotherapy or surgery can also affect the initial status of gonads before treatment starts. This will determine the final impact on ovarian function.

Cumulative doses of cyclophosphamide can cause infertility in young women [13]. Gonadotoxicity is directly related to age: the cumulative dose needed to cause premature ovarian failure decreases as age increases. Its effect can be acute or cumulative, and the ovarian capacity for recovering is limited. Chemotherapy and radiotherapy frequently induce a reduction in the number of germ cells, with a loss of steroid hormones, the possibility of mutation or teratogenic consequences [14].

The implications of chemotherapy treatment on fertility and future pregnancy has a higher relevance for younger women as most are childless or haven’t completed their family. But, as gonadotoxicity is an age-related process, their younger age will have a protective effect. Many younger patients will naturally recover their ovarian function and fertility, especially if the applied chemotherapy doses are low.

But regardless of chemotherapy, age itself is an important factor related to fertility decline; as ovarian reserve decreases with age at the time chromosomically abnormal embryos increase. It is well known that fertility significantly declines in patients over 35 years of age, and it is a fact that modern society forces a growing number of women of childbearing age to delay motherhood [15].

Chemotherapy

It is known that chemotherapeutic agents can cause mutations, DNA adducts and structural breaks, as well as oxidative damage in somatic and germ cells. Alkylating agents such as cyclophosphamide or ifosfamide are the most gonadotoxic agents, but also gonadotoxic are chlorambucil, busulfan, cisplatin, melphalan, carboplatin, or procarbazine [2, 12, 16].

The effect of chemotherapy on the ovary is not an all-or-nothing phenomenon, so the number of surviving primordial follicles following chemotherapy will depend on several factors such as age, type of agent and doses received [13].

Chemotherapy’s alkylating agents join with DNA, avoiding its replication and transcription [17]. They are extremely gonadotoxic by acting at any phase of the cellular cycle (cellular cycle phase independent), causing damage to the primary follicles. Pathological examinations of ovarian biopsies in patients treated with cyclophosphamide show either a total absence or a significant reduction in the number of inactive follicles, with fibrosis and no signs of follicular maturation [18].

The mechanism of chemotherapy causing premature ovarian failure is not well known, but granulosa cells appear to be the crucially affected cells [18]. Cellular edema of pre-granulosa cells is observed, with queratin deposits and edema of the nucleus of the cell, which damages the oocyte morphology [19]. Additional factors, such as vascular alterations and fibrosis of the ovarian cortex, may contribute to the reduction of follicles [20].

What is clear is that both the antral follicle count and ovarian volume decrease after chemotherapy. A fast fall in anti-Müllerian hormone (AMH) and inhibin B concentrations is observed during chemotherapy, although estradiol concentrations are maintained [21].

Radiotherapy

Similar to chemotherapy, the effect of radiotherapy on the gonads depends on age, cumulative doses, fractioned doses, and irradiation area. The average dose needed to destroy oocytes in humans is 2 Gy [22]. Ninety-seven percent of women receiving 5.0–10.5 Gy will subsequently undergo ovarian failure [23].

Especially if radiotherapy is applied during childhood, the irradiation area has been associated with alterations of the uterine function due to the reduction of vascular flow and endometrial thickness [24]. Cranial irradiation with 35–45 Gy doses can damage the hypothalamus–pituitary–gonadal axis, but, as gonads are not affected, they recover their function with gonadotropin replacement.

In males, the effect of radiotherapy will depend also on the dose, the fractionation scheme, and the individual susceptibility of each one, but permanent azoospermia is achieved with single doses >4–6 Gy, fractionated doses >1.2 Gy, or when total body irradiation is received in a Bone Marrow Transplant.

Surgery

Repetitive ovarian surgery due to endometriosis or another benign pathology may diminish the ovarian reserve and lead to premature ovarian failure. Furthermore, in recurrent endometriosis, normal residual ovarian tissue may be compromised.

Tubal sterilization through electrocoagulation, when compared to the application of mechanical clips, also seems to have an adverse effect on ovarian reserve in the postoperative period. Significant differences have been detected in ovarian volumes and antral follicle counts at 10 months after the tubal occlusion [25].

Treatment for Rheumatic Diseases

There are four main categories of drugs for the treatment of rheumatic diseases: (1) anti-inflammatory drugs, (2) corticosteroids, (3) immunosuppressive drugs, and (4) biological agents. These treatments are split into two main groups: the disease modifying antirheumatic drugs (DMARDs) and the nonsteroidal anti-inflammatory drugs (NSAIDs).

Disease Modifying Antirheumatic Drugs

DMARDs are a host of new drugs. Although most of the patients diagnosed with rheumatic diseases are treated with nonbiological DMARDs, the rate of biological DMARDs is increasing. The gonadotoxic effects of the anti-inflammatory and immunosuppressive drugs have not been studied with the exception of salazopyrine and some cytotoxic drugs as described here.

1. 
   

   – Salazopyrine impairs fertility in males, although not females, with a higher incidence of oligospermia, decreased sperm motility and higher rates of abnormal forms. Men with inflammatory bowel disease treated with salazopyrine showed a higher incidence of fetal abnormalities among offspring. Folate deficiency may have a role, as salazopyrine inhibits the gastrointestinal and cellular uptake of folate [26], but salazopyrine also has its own role as fetal abnormalities weren’t avoided with folate supplementation. An oxidative stress mechanism of male-induced infertility has also been described [27]. Usually, spermatogenesis recovers at about 2 months after withdrawal of the drug [28].

   
   
2. 
   

   – Cyclophosphamide and chlorambucil are rarely used in the treatment of rheumatoid arthritis, but these drugs are very important for patients with systemic lupus erythematosus. Cyclophosphamide is gonadotoxic in both sexes. It is not possible to predict which patients will become infertile and which will recover reproductive function, this depending fundamentally on age and the cumulative dose [28].

   
   
3. 
   

   – Methotrexate: There is a lack of evidence regarding effects of methotrexate on male fertility. The recommendation to stop methotrexate three months prior to conception is safe, but is not evidenced by an understanding of the impact of methotrexate on spermatogenesis or paternal-mediated teratogenicity but rather the timeframe of spermatogenesis. Given the unclear evidence, patients treated with methotrexate must be counseled on the likelihood of adverse effects of methotrexate and role of sperm cryopreservation [29].

Non-steroidal Anti-inflammatory Drugs

Inhibitors of cyclooxygenases (COX-1 and COX-2) are involved in ovulation and implantation. Transient infertility has been described after treatment with NSAIDs, such as indomethacin, diclofenac, piroxicam, and naproxen. NSAIDs can inhibit the rupture of the luteinized follicle and, thereby, cause transient infertility [30].

A decreased sperm count has been found in chronic male users of NSAIDs at low or moderate doses [30].

Fertility Preservation Procedures

Several strategies have been proposed to protect and preserve the ovarian function in patients with cancer or suffering from other pathologies with a high risk of premature ovarian failure. Some have demonstrated their efficiency and are now part of the daily routine of clinical practice, while others are still under evaluation.

These options include embryo and oocyte cryopreservation, cortical or whole ovary cryopreservation and GnRHa protection. IVM of immature oocytes still needs improvement, but there is no doubt that it will become an important part of these procedures in the future as the trend in fertility preservation techniques should be directed toward ovarian tissue cryopreservation and further retrieval of immature oocytes followed by IVM and vitrification [31].

Unlike with cancer patients for whom chemotherapy needs to be started immediately, these patients usually have no problem with the time frame of the 2–3 weeks needed to obtain the oocytes, as there is no hurry to complete the ovarian stimulation. Neither patients with endometriosis nor young people who wish to postpone childbearing are inconvenienced by this time frame. Indeed, some stimulations cycles can even be performed to increase the number of oocytes when oocyte vitrification is intended.

Oocyte Vitrification

Oocyte vitrification is a method of cryopreserving human oocytes which provides an excellent clinical outcome [6]. Vitrification is solidification of a solution by an extreme elevation of viscosity using high cooling rates, from −15,000 to −30,000°C/min, which avoids ice crystal formation and, thus, the damage and the osmotic effects caused by intracellular ice formation.

One of the problems of vitrification is the toxicity of cryoprotectants. This can be reduced by the use of an adequate combination of cryoprotectants (ethylene glycol + dimethylsulfoxide [DMSO] + sucrose) or by using very low volumes, which increases the speed of the vitrification process and consequently reduces the use of cryoprotectant in the vitrification solution [32].

The Cryotop method is a minimal volume device where oocytes are vitrified in volumes <0.1 µl, which preserves their capacity for fertilization and further development after warming. Survival rates >95% have been referred in young patients, although it is highly dependent to age and quality of the oocytes, and clinical results regarding fecundation and implantation rates, embryo quality, or pregnancy rates are similar to those obtained with fresh oocytes [33]. In addition, the number of pregnancies resulting from oocyte cryopreservation is constantly increasing, with no apparent increase in adverse postnatal outcome such as low birth weight or congenital abnormalities [34].

Regardless of the possible situations and the different methods available to preserve fertility, cryopreservation of oocytes can now be considered the elective method in noncancerous patients due to its reproducibility and effectiveness, in addition to the fact that surgery is not needed. No longer considered experimental, oocytes vitrification is an established, reproducible, safe, and effective technique according to the available evidence. But it is important to point out patients cryopreserving oocytes that pregnancy is not guaranteed after this procedure. It will just provide the opportunity to attempt an in vitro fertilization (IVF) cycle with the same prognosis the patient had in the moment she vitrified her oocytes, and with a limited number of attempts depending on the availability of oocytes.

Embryo Cryopreservation

Embryo cryopreservation is a widely accepted method that previously was considered the standard practice for fertility preservation [35], as thawed embryos were considered to achieve higher survival rates than oocytes. But the female patient needed to have a partner or use sperm donor to fertilize the retrieved oocytes, creating embryos that may not have been used in the future, which had various ethical considerations.

It has clearly been replaced nowadays by oocyte cryopreservation as it avoids the need for sperm at the time of oocyte retrieval and the results have been similar to embryo cryopreservation, and thus should be considered as the elective option.

Ovarian Tissue Cryopreservation

Ovarian tissue freezing for later autotransplantation is another alternative for fertility preservation. Immature oocytes in primordial follicles of the ovarian cortex are less sensitive to cryopreservation damage [36]. Thus, ovarian tissue freezing is an alternative to ovarian stimulation and oocyte cryopreservation for preserving fertility. Orthotopic transplantation of the frozen-thawed ovarian cortex would allow natural fertility and, in the case of failure, IVF would still remain an option. Another advantage of this approach, apart from future childbearing, is that patients would be able to restore ovarian function.

Ovarian cryopreservation and transplantation procedures have so far been almost exclusively limited to avascular cortical fragments. Transplantation of an intact ovary with vascular anastomosis has been proposed as a way to reduce the ischemic interval between transplantation and revascularization.

Despite an increasing number of successful reports of ovarian cortex transplantation (OCT) procedures [37–39], the safety of OCT is under evaluation since it is still considered an experimental technique. This is mainly due to the risk of reseeding cancer cells with the graft, especially for different tumors [40], although this risk shouldn’t be for non-cancer patients. Recently a few newborns have been obtained after OCT in leukemia patients, and that is why in Israel it stopped being considered experimental [41].

One of the main disadvantages of this technique is that it requires surgery – laparoscopy – to obtain the ovarian tissue and a further reimplant with appropriate incorporation of the cryoprotectant to the tissue. Ischemic damage and reduced follicular pool usually appear after transplantation. The active life of the transplanted tissue will depend on the neoangiogenesis and new vascularization.

Ovarian tissue cryopreservation is the main indication for prepubertal girls and in those situations with no available time to carry an ovarian stimulation [42].This approach can offer also great possibilities to patients, since portions of healthy tissue can be preserved for a further use when an oophorectomy is performed for a benign indication. The ethical basis for performing this surgery for elective cryopreservation has been discussed [43], but a patient’s request for cryopreserving small portions of ovary at the time of any other gynecologic surgery should not be denied on ethical grounds.

Gonadal Medical Protection

In vitro Maturation

Immature egg retrieval for further in vitro oocyte maturation and vitrification is a promising option for the future but still regarded as experimental. Most of the follicles in human ovaries remain primordial. Thus, they would be the most abundant source of oocytes but, due to their immaturity, IVM is needed. Primordial follicles can be isolated from either fresh or cryopreserved ovarian tissues and matured in vitro for further vitrification.

Although many healthy children with normal outcomes have been born after fertilization of fresh IVM oocytes, very few live births have been reported with the use of IVM oocytes that have been cryopreserved and thawed or warmed [9].

The main benefit is the absence of stimulation and its low cost, but results are not consistent enough and still need to be improved. Pregnancy and implantation rates are lower than those obtained with standard IVF cycles [50,51], and a higher clinical miscarriage rate has been observed [52]. So, more controlled studies are needed of the possible long-term effects of IVM on babies.

This method may be considered for patients in whom hormonal ovarian stimulation is not recommended due to high estradiol levels, such as breast cancer patients or those suffering from systemic lupus erythematosus. It may also be suitable for patients with polycystic ovary syndrome (PCOS), when there is an urgent need to start cytotoxic therapy or for prepubertal girls who cannot undergo ovarian stimulation.

Transposition of Ovaries

Scatter radiotherapy can cause considerable damage even if the gonads are outside the radiation field. The purpose of this approach is to avoid the direct exposure of the ovaries to radiotherapy, although the indirect exposure can also cause gonadotoxicity. Thus, it should be indicated for any pathology that requires pelvic radiotherapy treatment. When this approach is performed, 16–90% of the patients show the ovarian function preserved [53].

Ovarian transposition is not suitable for non-oncological patients as radiotherapy is uncommonly used, although it is very useful for cancer patients when they are going to receive local radiotherapy.