Main results and possible practical consequences
In the present study, ovarian function of rats was investigated with a stepwise reduction of the total ovarian tissue from 0% (group 1) to 25%, 50%, 75%, 82.5% and 100% in groups 2,3,4,5,6, respectively. The most remarkable result is that excision of up to 75% of total ovarian tissue will still allow normal production of E2 and P. To our knowledge, has been shown for the first time in an animal model.
If this is also would be true for women undergoing removal of ovarian tissue for pathological reasons, e.g. ovarian endometriosis, in patients with cancer or to remove enough tissue for cryopreservation, the consequence would be that despite only 25% tissue remaining, HRT for treatment of estrogen-deficiency consequences such as climacteric complaints or osteoporosis would not be necessary. Possible detrimental HRT risks like breast cancer, thromboembolism and stroke could therefore be avoided . This is important when using OTC/OTCT for fertility protection, also for women not wanting fertility but with POI for other reasons [15 − 18,20].
Hormone levels for assessment of ovarian function
We found in our study that removal of < 75% of the total ovarian tissue (group 2,3,4) maintains normal production of E2 and P (i.e. no significant difference to control), whereas FSH was significantly increased after 75% tissue excision (i.e. in group 4,5 and 6). It is well known that despite the negative feedback mechanisms within the HPO axis, E2 and P values can remain normal but FSH can increase, even in cases of higher E2 levels, especially during menopausal transition and often together with a decrease in INHB [24 − 26] .
From the AMH levels it can be suggested that in all experimental groups (2 to 6) there was a decrease in ovarian reserve directly after surgery. It has been suggested that serum AMH levels show the greatest sensitivity to ovarian insufficiency, particularly a strong correlation with the number of early antral follicles. This can explain the early decrease in AMH after excision of only 25% of total ovarian tissue. The decrease in INHB was later (starting 6 weeks after surgery) compared to AMH (starting 2 weeks after surgery) and was only seen if 75% and more of the total ovarian tissue was removed, i.e. in group 4,5 and 6. Considering these results with INHB, the decrease is larger with the time after surgery and, as expected, with the amount of tissue removal.
We can conclude that ovarian reserve is starting to be significantly decreased after 75% removal, i.e. in group 4,5 and 6. However, E2 and P are still produced in group 4 with normal values. This hormonal constellation can be clinically compared with women during hormonal transition where even pregnancy is possible with occasional ovulation and young women with a similar hormonal constellation (for example POI) can also become pregnant .
If fertility could be maintained after up to 75% excision of total ovarian tissue (which needs further research), our animal data strongly suggest that HRT can be avoided if also shown in human studies. In context with very new data investigating breast cancer risk during HRT use, which is mostly feared by patients and doctors, this result could be important. According to a recent meta-analysis including 58 studies , the number of additional breast cancer patients when using HRT is about five fold higher than calculated from earlier studies, with the striking result that even only one year of treatment with HRT can significantly increase breast cancer risk. This is especially important for OTC/OTCT, because for most patients retransplantation is only possible one or two even more years after cryopreservation.
Ovaries’ volume and morphology
There was no significant difference in ovarian volume between group 3 and control. However, this may be due to the short observation time. This assessment was of interest because a compensatory increase in volume of the remaining ovary is known for women after unilateral oophorectomy .
No morphological differences in the follicles were seen in group 2–5 12 weeks after surgery compared with the control (Fig. 4). Because of the different ovarian volumes, the size of ovarian tissue sections is inconsistent and follicle count cannot be performed, but a follicular form can be seen in each group. This is well understood, as Jacques Donnez et al. Mentioned  that ovarian tissue extraction surgery itself does not cause damage to the follicles. The difference in hormone levels in our experiments is perhaps mainly due to the difference in volume of the remaining ovaries, as different ovarian volumes can have clinical implications in ovarian activity .
Should unilateral oophorectomy be recommended?
Unilateral oophorectomy is often performed in clinical practice. In our animal model, i.e. removal of 50% of total ovarian tissue in group 3, secretion of E2, P, FSH, AMH, INHB was not affected. Clinical studies have shown a rather weak association of unilateral oophorectomy with ovarian endocrine and reserve function which was explained by compensatory growth of the remaining ovary [29,31 − 32]. It has been suggested that new primordial follicles may be generated from germline stem cells and oocytes develop from primitive germline cells harvested from women of reproductive age . Normal values of FSH and INHB, as seen in group 3, may also be explained by greater feedback sensitivity in the HPO-axis of the remaining ovary.
Some studies suggest that unilateral oophorectomy is effective in restoring long term ovulation function in PCOS patients and after treatment of cancer [34,35]. It is reassuring that women with only one ovary do not seem to have a reduced potential to conceive, either naturally or via IVF . However, the outcomes of unilateral oophorectomy may differ in these populations and removing one ovary can be a strong risk factor for early menopause , particularly before the age of 35 years. It can adversely affect the fertility potential of women who already have reduced ovarian reserve and multiple risk factors for early menopause . Despite our encouraging experimental results, further research is needed for these patients, and unilateral oophorectomy should not be currently recommended.
Changes in Weight
Among the five experimental groups, only the bilateral oophorectomy group (group 6) showed significant changes in weight - a significant gain compared to control (Table 1). This is consistent with clinical findings in women with weight increase after menopause . This may be due to decreased estrogen production causing changes in metabolic function (deterioration in lipid and glucose metabolism) with changes in the energy budget. In our study, all rats in group 6 were acutely postmenopausal because of "surgical menopause". Increased weight was also observed in group 5, but was not significant. We speculate that this was not because of 17.5% ovarian tissue remaining, but because they were postmenopausal since E2 and P significantly decreased and FSH increased, comparable to group 6.
Strength and Limitations
This study is the first to determine the effects of stepwise removal of ovarian tissue on endocrine and reserve function in rats. It may have been possible to obtain more information with more groups, e.g. testing 32.5% ovarian tissue. However, after discussion with our ethical committee, we wanted to limit the number of animals used, and we doubt that more relevant information would have been obtained by testing more groups. And we designed 6 rats in each group according to the suggestions from the experts of ethical committee. We investigated the early stage impact of stepwise removed ovarian tissue, so we only observed 12 weeks after surgery. Further long-term observation of ovarian function could be of interest, especially for group 4, where we could expect that not only the production of E2 and P is maintained despite of 75% removal of total ovarian tissue, but other parameters could recover, also leading to maintenance of fertility.
Summary and Conclusion
Excision of ovarian tissue is necessary for women wanting cryopreservation and later retransplantation to preserve fertility and for pathological changes in the ovaries. Function of the remaining tissue is wanted because retransplantation of tissue may not be successful. HRT to treat and prevent estrogen deficiency sequelae is often rejected because of potential risks or may not be possible due to contraindications.
Our animal study confirms that removal of one ovary does not affect the secretion of E2, P, FSH, AMH and INHB, i.e. the function of ovarian tissue is maintained. However, to our knowledge we have demonstrated for the first time that even removal of up to 75% of the total ovarian tissue can still maintain normal production of E2 and P. If confirmed in women, risky HRT could be avoided. It may be appropriate in the future to increase the excision volume of ovarian tissue for cryoconservation to achieve enough ovarian potency after retransplantation, since chemotherapy can lead to severe POI. Further research is needed to investigate if not only E2/P production, but also fertility can be maintained with only 25% remaining total ovarian tissue.