In Vitro Maturation of Oocytes Obtained from Ovarian Cortex Among Postpubertal Hematological Cancer Patients Undergoing Fertility Preservation

Abstract

Purpose:

In vitro maturation (IVM) of oocytes obtained from ovarian tissue during ovarian tissue cryopreservation (OTC) is a technique for fertility preservation in patients with cancer obviating the need to postpone chemotherapy initiation. Little is known about IVM outcomes in hematological malignancies, especially post-chemotherapy. The purpose of this study was to evaluate the effect of cytotoxic treatment on the potential to retrieve immature oocytes and mature them in vitro and examine the association between serum inflammatory markers and these results.

Methods:

In this retrospective study, we evaluated inflammation markers, including B symptoms and IVM outcomes of 78 chemotherapy-naive and exposed patients diagnosed with Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), or acute myeloid leukemia (AML).

Results:

The mean number of oocytes found was 7.2 ± 7.2. The average number of oocytes matured by IVM was 2.8 ± 3.5, and a mean IVM rate was 32.1 ± 27.7%. All patients in the ALL and AML groups had previous exposure to chemotherapy before OTC, compared with 50.0% (7/14) and 31.9% (15/47) in the NHL and HL groups, respectively. Among patients with lymphoma, chemotherapy exposure was associated with the reduced number of retrieved oocytes (9.8 ± 7.7 vs. 5.3 ± 5.7 oocytes, p = 0.049) in the HL group but not with the number of mature oocytes or IVM rate. B symptoms were not associated with IVM outcomes. Lymphocyte count (ß = 1.584; p = 0.038) and lactate dehydrogenase (ß = 0.009; p = 0.043) were the only significant parameters associated with the number of matured oocytes in a linear regression model.

Conclusion:

IVM is a promising assisted reproductive technology, which holds great potential for patients in need of urgent fertility preservation or those who cannot receive hormonal stimulation. Our results demonstrate the feasibility of the technique even in the presence of B symptoms and elevated inflammation markers and in patients with previous exposure to chemotherapy.

Introduction

The incidence of malignant diseases and hematological malignancies, in particular, has been steadily increasing in recent decades.¹ Overall, among children and adolescents in the United States, the most common types of cancer are leukemias, brain and central nervous system tumors, and lymphomas.¹

When referring to U.S. data, during the years 1999–2014, the cancer death rate for children and adolescents aged 1–19 years declined by 20%² and more dramatically in acute lymphoblastic leukemia, the most common childhood cancer.^3,4 This trend is also reflected in the increasing number of young women with hematological malignancies who seek the option of fertility preservation (FP) while facing chemotherapy regimens.

The main treatment options offered to patients with cancer include chemotherapy, radiation, and bone marrow transplantation (BMT) in hematological cases. These life-saving treatments encompass a risk of significant gonadotoxic effects on ovarian tissue and follicular reserve.⁵ The degree of this cytotoxic effect is correlated with the chemotherapy regimen, the overall dose, and the patient’s age.⁶

FP for patients with cancer includes cryopreservation of mature oocytes aspirated under sonography after hormonal stimulation, freezing of embryos, and cryopreservation of ovarian cortical tissue (OTC) with an option of future auto-transplantation to restore hormonal activity and fertility.^7–10 In vitro maturation (IVM) in the context of FP during OTC includes aspiration of immature oocytes from the antral follicles observed on the ovarian tissue surface together with oocytes found in the media after tissue processing and following OTC.⁹ Oocytes retrieved are incubated in a designated medium for 24–48 hours to induce maturation from the germinal vesicle stage to the mature oocyte—the metaphase II (MII) stage. The ability to induce maturation of immature oocytes in vitro and cryopreserve them as mature oocytes available for future fertilization provides an additional option for FP. IVM is a valid option in both pre- and postpubertal patients, and a combined approach of cryopreservation of ovarian tissue and IVM of germinal vesicle oocytes retrieved during the tissue dissection (ovarian tissue oocyte IVM [OTO-IVM]) provides an additional FP option,^11–14 even in cases of patients with gynecological cancer (advanced endometrial or ovarian cancer) undergoing radical surgery.¹⁵

Moreover, although recent studies showed that careful evaluation of the ovarian tissue before transplantation allows a safe transplantation option in leukemia survivors who are in complete remission stage at the time of OTC,^16,17 leukemia survivors are sometimes advised to avoid auto-transplantation of ovarian tissue to restore fertility, because of the concern that the transplanted tissue may risk them with reseeding of malignant cells and reimplantation of cancerous cells,^18–20 and in such cases the importance of the IVM process, which allows cryopreservation of a single, noncancerous cell for future fertility, is emphasized.

In patients opting to preserve fertility because of malignant disease, it is preferable to perform the procedure before the initiation of cytotoxic treatment,²¹ though recent studies have shown that prior chemotherapy does not impair ovarian tissue auto-transplantation success.¹⁸ Occasionally, there is a need to perform it after receiving chemotherapy and before further cytotoxic treatment that may cause additional and more severe damage to the ovarian reserve. Current literature regarding the effect of cytotoxic treatments on IVM potential is scarce, and the significance of the timing and the extent of chemotherapy exposure on the IVM process remains unknown.

Interestingly, the malignancy itself has been demonstrated to affect oocyte growth and maturation.²² One of the hypotheses proposed to explain this finding is the effect of systemic inflammatory processes present in patients with cancer. To the best of our knowledge, no previous study has evaluated the association between systemic inflammation markers and the IVM potential of oocytes retrieved in patients with cancer.

In this study we aimed to focus on the effect of cytotoxic treatment in adolescents and young women diagnosed with hematological cancer, on the potential to retrieve immature oocytes and then mature them in vitro. Furthermore, we examined the association between serum inflammatory markers and the results of retrieval of oocytes and their maturation potential.

Materials and Methods

Patients

Our study cohort consisted of postpubertal patients aged 12–32 years who were diagnosed with Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), or acute myeloid leukemia (AML). All patients underwent OTC between 2002 and 2021 and had an attempt of retrieval of immature oocytes from the ovarian tissue or medium for IVM. Both chemotherapy-naive and exposed patients at the time of OTC and IVM were included in this study.

Patients under 18 years were consulted regarding FP options with their parents or guardians together with a pediatric hematology/oncology specialist. Patients who were interested in preserving their fertility by cryopreservation of ovarian cortex tissue were referred for a laparoscopic oophorectomy for OTC after signing an informed consent.

The electronic records of patients included in the study, as well as the IVF laboratory database, were scanned. We collected demographic, oncologic, reproductive, and gynecological data. Information regarding the chemotherapy regimen and timing with regard to OTC and hormonal profile (when available) was collected. Additional information regarding the OTC procedure and IVM outcome was collected and included the number of ovarian tissue ampules cryopreserved (approximately five tissue strips in each), the number and stage of oocytes retrieved from the medium—number of immature oocytes, the number of oocytes matured using IVM, and the IVM rate per patient. Laboratory tests commonly used in the assessment and prognosis of lymphoma and leukemia in females and males^23–26 were also collected—albumin (g/L), lactate dehydrogenase (LDH) levels (U/L), hemoglobin (g/L), and the presence of B symptoms. Systemic B symptoms were defined as at least one of these three symptoms—night sweats, fever >38°C, or weight loss >10% body weight over a period of 6 months. In addition, platelets (10³/µL), white blood cells (WBC, 10³/µL), and absolute lymphocyte counts (10³/µL) were collected.

Specimen handling and IVM process

Complete or partial (roughly two-thirds) oophorectomy was performed in a laparoscopic approach under general anesthesia. Following the removal of the ovary, the tissue was placed on ice in a vial containing Leibovitz L-15 medium (GIBCO-BRL). The tissue was transferred immediately to an adjacent IVF lab for processing and dissection.²⁷ The ovarian cortex was dissected from the ovarian medulla and cut into 5–10 mm³ (5 × 1 × 1–2 mm) strips inside the medium. The cortex strips (besides for one that was sent to pathology) were transferred to a 1.5 M dimethyl sulfoxide and 0.1 M sucrose precooled freezing medium.

One to five ovarian cortex slices were placed in a 2 mL cryovial that contained a cryoprotectant medium and cryopreserved in a programmable freezing machine (Kryo 360) using a slow freezing protocol. Liquid nitrogen was used for storage of the frozen vials. The media in which we dissected the tissue and the ovarian cortex (following the dissection of the tissue) were scanned for the presence of cumulus–oocyte complexes under the microscope.

A dissecting microscope was used to aspirate antral follicles observed on the ovarian surface. The oocytes retrieved from the ovarian surface and the oocytes found in the media were flushed with a designated medium and incubated as previously described.¹⁰

All germinal vesicle (GV) oocytes collected were subjected to an IVM process using a Sage media (Al-Rad Medical), supplemented with 0.075 IU/mL luteinizing hormone (LH) and 0.075 IU/mL follicle-stimulating hormone (FSH).

We incubated cumulus–oocyte complexes in a SAGE IVM culture media, and 24 hours later, denudation of the oocytes was performed and the oocytes were assessed for maturation. Immature oocytes were incubated in a fresh culture media, followed by a second evaluation 24 hours later. Oocytes that did not reach the MII stage were discarded, while the matured MII oocytes were cryopreserved using slow freezing or vitrification for future use.

Chemotherapy regimens and previous exposure

Chemotherapy agent regimens received by patients in our cohort were based on chemotherapy protocols that patients were enrolled on (risk for chemotherapy gonadotoxicity was previously described^28,29): (1) adriamycin, bleomycin sulfate, vinblastine sulfate, and dacarbazine; (2) rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine and prednisone (R-CHOP); (3) daunorubicin and cytarabine; (4) dactinomycin, vincristine, etoposide, prednisone, and adriamycin, and (5) additional regimens that include alkylating agents (other than R-CHOP). The age groups for this study were (1) menarche to 17 years, (2) 18–24 years, and (3) 25 years or older.

We analyzed OTC outcomes (number of ovarian tissue ampules cryopreserved and number of immature oocytes retrieved) and IVM process outcomes (number of oocytes in vitro matured and the IVM rate) according to tumor type groups and by factors with potentially harmful effects on outcomes—previous chemotherapy exposure and presence of B symptoms before OTC.

The study conforms to the provisions of the Declaration of Helsinki and was approved by the Human Research Ethics Committees of the Hadassah University Hospital (IRB 0288-16-HMO).

Statistical analysis

Categorical variables were assessed by the chi-square test and Fisher’s exact test (applied when >20% of cells have expected frequencies of less than five). Continuous variables were compared by the Student’s t-test or Mann–Whitney U test (for non-normally distributed parameters) and presented as mean ± standard deviation (SD) or median and interquartile range (IQR). The comparison between independent continuous variables of the four cancer-type groups was performed using the one-way analysis of variance (ANOVA) or the nonparametric Kruskal–Wallis test (for non-normally distributed parameters) followed by the post hoc Tukey’s Honest Significant Difference test for multiple comparisons. The cross-sectional association of patients’ parameters and IVM rate was tested using the Pearson correlation coefficient (r). ANOVA was performed to evaluate the association between patients’ characteristics and the number of matured oocytes and the IVM rate. No comparisons were conducted between the chemotherapy groups because of the limited number of patients who were additionally divided into four groups. Tests were two-tailed with a p value of <0.05 considered significant for all comparisons, and the statistical analysis was performed using SPSS software (version 22; IBM Corp.).

Results

Overall, 78 patients with leukemia and lymphoma aged 12–32 years (mean age of 21.3 ± 5.8, median 20.0 years) underwent an OTC procedure in our FP center between 2002 and 2021. The average number of ovarian tissue ampules cryopreserved was 12.7 ± 3.4, and the number of retrieved oocytes was 7.2 ± 7.2. For patients who had oocytes retrieved (66/78 [84.6%] patients), the average number of oocytes matured by IVM was 2.8 ± 3.5, and the mean IVM rate was 32.1 ± 27.7%.

Sixty-one out of the 78 patients were patients with lymphoma: 47 of them had HL and 14 had NHL. The leukemia group (n = 17) was smaller and consisted of seven women with ALL and 10 women with an AML diagnosis.

Table 1 presents the basic characteristics, hormone levels, blood count parameters, LDH, C-reactive protein, and albumin levels for each of the four groups—HL, NHL, ALL, and AML.

Table 1.

Basic Characteristics of the Four Tumor Type Groups at the Time of Ovarian Tissue Cryopreservation for Fertility Preservation

Parameter	Hodgkinlymphoma	Non-Hodgkinlymphoma	Acute lymphoblasticleukemia	Acute myeloidleukemia	p-Value
Number of patients	47	14	7	10
Age (years)	20.0 ± 4.8^*	24.2 ± 5.5^*	19.9 ± 7.3	24.2 ± 7.4	0.031
BMI (kg/m²)^a	23.0, 5.8 (12)	24.0, 5.2 (7)	23.7, N/A (2)	23.1, 4.1 (5)	0.952
Chemotherapy before OTC	15/47 (31.9%)	7/14 (50%)^**	7/7 (100%)	10/10 (100%)	<0.001
B symptoms before OTC^b	10/33 (30.3%)	3/7 (42.8%)	—	3/4 (75.0%)	0.208
Hormone levels pre-OTC^c
FSH (maul/mL)^a	5.8, 4.8 (19)	2.1, 2.4 (5)	6.6, 6.0 (7)	5.6, 4.7 (7)	0.124
LH (mIU/mL)^a	4.0, 6.9 (19)	1.9, 3.2 (5)	2.3, 8.4 (6)	1.0, 13.7 (8)	0.443
Estradiol (pmol/L)^a	290, 416 (15)	271, N/A (3)	108, 908 (5)	69, 43.7 (4)	0.413
AMH (ng/mL)^a	1.3, 2.4 (6)	0.0, N/A (3)	0.4, N/A (1)	—	0.097
Complete blood count
WBC (10³/µL)	10.6 ± 4.4^*	6.9 ± 3.5^*	5.0 ± 2.9^*	9.1 ± 8.3	0.009
Lymphocytes (10³/µL)^a	1.5, 1.2 (46)	1.0, 0.5 (13)	0.9, 0.5 (7)	1.1, 0.5 (9)	0.13
Lymphocytes (%)	15.7 ± 8.0^***	17.7 ± 10.2	27.3 ± 12.7^***	19.6 ± 7.6	0.016
Hemoglobin (g/L)	11.1 ± 1.4	11.5 ± 1.6	10.7 ± 1.9	9.9 ± 1.9	0.108
Platelets (10³/µL)	301.1 ± 111.5	268.2 ± 101.7	253.7 ± 84.2	210.4 ± 100.9	0.112
LDH (U/L)^d	420.7 ± 182.2	431.5 ± 211.9	457.7 ± 266.0	488.3 ± 85.1	0.805
Albumin (g/L)^d	40.7 ± 4.9	42.1 ± 4.7	40.4 ± 35.9	40.0 ± 4.9	0.796

Data presented as mean ± SD, median, IQR (number of patients with measurement), or n/N (%).

Non-normally-distributed variable. Kruskal–Wallis test was performed and the result presented as median (N).

B symptoms were defined as at least one of these three symptoms: night sweats, fever >38°C, or weight loss >10% body weight over a period of 6 months. Data regarding B symptoms’ presence or absence were available for 33/47 patients with Hodgkin lymphoma, 7/14 patients with non-Hodgkin lymphoma, and 4/10 patients with acute myeloid leukemia.

Baseline FSH, LH, and estradiol levels measured by hormone panel test on day 3 of the menstrual period.

Tested on serum samples.

Post hoc Tukey’s Honest Significant Difference (HSD) test showed a significant difference (p < 0.05) between the Hodgkin lymphoma group and non-Hodgkin lymphoma and acute lymphoblastic leukemia groups.

p < 0.05 when compared with the acute myeloid leukemia group.

***

Post hoc Tukey HSD test showed a significant difference (p < 0.05) between the Hodgkin lymphoma group and acute lymphoblastic leukemia.

AMH, anti-Mullerian hormone; BMI, body mass index; FSH, follicle-stimulating hormone; IQR, interquartile range; LDH, lactate dehydrogenase; LH, luteinizing hormone; OTC, ovarian tissue cryopreservation; SD, standard deviation; WBC, white blood count.

The groups differed in age (p = 0.031), WBC level (p = 0.009), and percent of lymphocytes (p = 0.016).

All patients in the ALL and AML groups had previous exposure to chemotherapy, whereas the NHL and HL groups had 50.0% (7/14) and 31.9% (15/47) of patients, respectively, with exposure to chemotherapy before OTC (Table 1). The number and frequency of patients previously exposed to chemotherapy based on the chemotherapy received are presented in Supplementary Table S1. Ovarian tissue cryopreservation outcomes—the number of ampules that contain ovarian tissue strips, the number of oocytes retrieved from the cortex and media (in which the tissue was dissected in), and the number of retrieved and the number of mature oocytes—did not differ between the four groups (Table 2). The percent of patients who had at least one oocyte retrieved and the IVM rate (%) were similar in the four groups. The only difference noted was in the rate of patients with successful IVM (at least one mature oocyte using IVM)—with 81% (34/42), 70% (7/10), and 83% (5/6) of patients with successful IVM in the HL, NHL, and ALL groups (respectively), compared with only 25% (2/8) in the AML group (p = 0.014).

Table 2.

Ovarian Tissue Cryopreservation and In Vitro Maturation Outcomes According to Tumor Type Groups

Parameter	Hodgkinlymphoma	Non-Hodgkinlymphoma	Acute lymphoblasticleukemia	Acute myeloidleukemia	p-Value
No. of patients	47	14	7	10
No. of tissue ampules cryopreserved	12.8 ± 3.0	13.1 ± 4.3	10.4 ± 3.4	13.2 ± 3.5	0.318
No. of immature oocytes retrieved	8.4 ± 7.3	7.2 ± 8.4	4.6 ± 5.9	3.7 ± 4.2	0.208
Patients with oocytes retrieved	42/47 (89.4%)	10/14 (71.4%)	6/7 (85.7%)	8/10 (80%)	0.348
No. of in vitro matured oocytes	3.3 ± 3.4	3.9 ± 5.3	1.2 ± 0.8	1.5 ± 2.9	0.281
Patients with oocytes matured	34/42 (81%)^*	7/10 (70%)	5/6 (83%)*	2/8 (25%)	0.014
In vitro maturation rate	34.0 ± 24.0%	31.8 ± 30.1%	44.2 ± 44.4%	13.2 ± 25.0	0.168

Data presented as mean ± SD or n/N (%).

p < 0.05 when compared with the acute myeloid leukemia group.

We additionally compared OTC and IVM outcomes for the HL group and the NHL group separately, by allocation to the following groups: exposed or nonexposed to chemotherapy and the presence or absence of B symptoms (Table 3). In the HL group, a comparison between patients who were chemotherapy naive (n = 32) and those exposed to chemotherapy before OTC (n = 15) showed a significant difference only in the ability to retrieve immature oocytes, which was improved in chemotherapy-naive patients (9.8 ± 7.7 vs. 5.3 ± 5.7 oocytes; p = 0.049). A similar comparison according to the presence of B symptoms at the time of OTC (10 patients had B symptoms and 23 did not) did not demonstrate a difference in outcomes.

Table 3.

A Comparison of Ovarian Tissue Cryopreservation and In Vitro Maturation Outcomes in Patients with Hodgkin and non-Hodgkin Lymphoma by Risk Factors

Parameter	Patients with Hodgkin lymphoma		p-Value
Parameter	Chemotherapy exposed (n = 15)	Chemotherapy naive (n = 32)	p-Value
No. of tissue ampules cryopreserved	12.8 ± 3.8	12.8 ± 2.7	0.985
No. of immature oocytes retrieved	5.3 ± 5.7	9.8 ± 7.7	0.049
Patients with oocytes retrieved	12 (80%)	30 (93.8%)	0.309
No. of in vitro matured oocytes	2.3 ± 2.6	3.7 ± 3.7	0.238
Median (IQR)^*	2.0 (4.0)	2.5 (4.0)
Patients with oocytes matured	8 (66%)	26 (87%)	0.127
In vitro maturation rate	31.4 ± 26.6%	35.0 ± 23.3%	0.663

	B symptoms (n = 10)^a	No B symptoms (n = 23)^a	p-Value
No. of tissue ampules cryopreserved	12.9 ± 2.2	12.4 ± 2.7	0.638
No. of immature oocytes retrieved	7.6 ± 7.1	8.4 ± 8.0	0.778
Patients with oocytes retrieved	10 (100%)	19 (82%)	0.289
No. of in vitro matured oocytes	3.2 ± 3.5	3.3 ± 3.1	0.961
Median (IQR)^*	2.0 (5.0)	2.0 (3.0)
Patients with oocytes matured	8/10 (80%)	17/19 (89%)	0.285
In vitro maturation rate	39.6 ± 22.6%	29.8 ± 18.9%	0.223

	Patients with non-Hodgkin lymphoma
	Chemotherapy exposed (n = 7)	Chemotherapy naive (n = 7)	p-Value
No. of tissue ampules cryopreserved	12.9 ± 5.5	13.3 ± 3.1	0.861
No. of immature oocytes retrieved	4.0 ± 4.1	10.4 ± 10.5	0.159
Patients with oocytes retrieved	5/7 (71%)	5/7 (71%)	1.000
No. of in vitro matured oocytes	1.8 ± 1.3	6.0 ± 7.2	0.234
Patients with oocytes matured	4/5 (80%)	3/5 (60%)	0.788
In vitro maturation rate	36.3 ± 33.1%	27.3 ± 29.9%	0.665

	B symptoms (n = 3)^a	No B symptoms (n = 4)^a	p-Value
No. of tissue ampules cryopreserved	12.0 ± 3.0	10.8 ± 3.9	0.668
Median (IQR)^*	12.0 (N/A)	12.0 (7.0)
No. of immature oocytes retrieved	11.3 ± 11.5	9.3 ± 11.0	0.817
Patients with oocytes retrieved	2/3 (66%)	3/4 (75%)	0.800
No. of in vitro matured oocytes	9.5 ± 9.2	5.0 ± 5.3	0.523
Patients with oocytes matured	2/2 (100%)	3/3 (100%)	1.000
In vitro maturation rate	48.4 ± 29.9%	43.8 ± 31.3%	0.881

Data presented as mean ± SD or n/N (%).

Calculated for non-normally distributed parameters.

Data regarding B symptoms’ presence or absence were available for 33/47 patients with Hodgkin lymphoma and 7/14 patients with non-Hodgkin lymphoma.

In the NHL population, the same comparisons, by groups of chemotherapy exposure and B symptoms, showed similar OTC and IVM results, though these subgroups were small (Table 3).

We conducted additional analyses comparing patients who received the R-CHOP protocol or other protocols containing alkylating agents (n = 14) with those who received chemotherapy not containing alkylating agents (n = 25). This comparison did not reveal a significant difference between the alkylating group and the nonalkylating group in terms of the number of preserved ampules (11.4 ± 3.3 vs. 13.1 ± 4.3 ampules; p = 0.185), the number of retrieved oocytes (4.1 ± 5.0 vs. 4.8 ± 5.1 oocytes; p = 0.717), and the number of in vitro matured oocytes (1.3 ± 1.1 vs. 2.1 ± 2.7 mature oocytes), while the age at OTC was similar between the groups, with 23.7 ± 6.8 years in the alkylating group and 22.4 ± 6.1 years in the nonalkylating group (p = 0.563).

Linear regression analysis was used to evaluate the association between patients’ characteristics (malignancy type, age, prior exposure to chemotherapy, B symptoms, laboratory data (albumin, hemoglobin, WBC, lymphocytes, platelets, and LDH levels) and the dependent parameter of the number of mature oocytes (Table 4). This analysis showed that the only two parameters positively associated with the dependent parameter of the number of in vitro matured oocytes are lymphocyte count (ß coefficient = 1.584, p = 0.038) and LDH levels (ß coefficient = 0.009, p = 0.043).

Table 4.

Linear Regression Model Evaluating the Association Between Patients’ Characteristics and the Number of Matured Oocytes

Parameter	ß coefficient	SE	95% CI	p-Value
Malignancy type
HL	−8.984	8.037	−25.698 to 7.729	0.276
NHL	3.751	2.02	−0.449 to 7.952	0.077
AML	−2.426	3.068	−8.807 to 3.955	0.438
Age	−0.074	0.137	−0.359 to 0.21	0.591
Previous chemotherapy exposure	1.768	1.41	−1.163 to 4.7	0.223
Systemic B symptoms	−2.105	1.772	−5.789 to 1.579	0.248
Albumin (g/L)	0.152	0.134	−0.128 to 0.432	0.271
Hemoglobin (g/L)	0.407	0.458	−0.545 to 1.36	0.384
WBC (10³/µL)	−0.09	0.175	−0.455 to 0.275	0.615
Lymphocytes (10³/µL)	1.584	0.716	0.09 to 3.073	0.038
Platelets (10³/µL)	0	0.006	−0.014 to 0.013	0.967
LDH (U/L)	0.009	0.004	0 to 0.018	0.043

AML, acute myeloid leukemia; CI, confidence interval; HL, Hodgkin lymphoma; NHL, non-Hodgkin lymphoma; SE, standard error.

An additional linear regression analysis, performed for the association between the above patients’ characteristics and the dependent parameter of IVM rate, did not find specific parameters associated with IVM rate, although the association between lymphocyte count and IVM rate was nearly significant (p = 0.058).

We additionally tested the correlation between IVM rate and several inflammation-related markers (Table 5)—albumin and LDH levels, hemoglobin, WBC, lymphocytes, and platelet count. In the entire cohort, the only parameter that had a significant correlation with IVM rate was LDH, with a linear correlation (r) of 0.36 (p = 0.005), representing a weak positive correlation.

Table 5.

Linear Correlation Evaluation Between IVM Rate and Inflammation-Related Markers in the Entire Study Population

Parameter	IVM rate
Parameter	N	Pearsoncorrelation (r)	p-Value
Albumin (g/L)	59	0.165	0.212
Hemoglobin (g/L)	64	0.097	0.444
WBC (10³/µL)	64	0.064	0.615
Lymphocytes (10³/µL)	64	0.215	0.088
Platelets (10³/µL)	64	0.070	0.581
LDH (U/L)	59	0.361	0.005

IVM, in vitro maturation.

Discussion

In this study we investigated the unique population of patients with lymphoma and leukemia undergoing FP by OTC and attempting to preserve in vitro matured oocytes obtained from the tissue and the medium in which it was dissected. This study is the first to investigate the correlation between cancer-associated inflammation markers and FP outcomes.

We were able to show that IVM is indeed a valid FP option in post-menarche hematological malignancies, both lymphoma and leukemia. Although this technique shows promise, comprehensive studies regarding its impact on reproductive outcomes are still lacking. It is assumed that pregnancy outcomes per in vitro matured oocyte may be notably low. When testing the linear correlation of inflammation-related markers and IVM rate, LDH levels were shown to have a positive, though weak, correlation with IVM rate. Moreover, both LDH and lymphocyte counts were positively associated with the number of in vitro matured oocytes in a regression model, though the ß coefficient was relatively low.

We additionally report on our findings regarding patients with cancer previously exposed to chemotherapy who later faced additional potentially gonadotoxic chemotherapy and decided to preserve ovarian tissue and use IVM on immature oocytes recovered. We show similar OTC and IVM outcomes in chemotherapy-naive and exposed patients, with the exception of patients with HL, in which the ability to retrieve immature oocytes was poorer in chemotherapy-exposed patients compared with those who did not receive chemotherapy. Similar to our HL cohort, in a recent study, Prades et al.³⁰ explored predictive patient characteristics associated with successful isolated immature oocyte retrieval in a cohort of 257 patients and found that rates of successful retrieval of isolated immature oocytes were significantly higher in patients who were not exposed to chemotherapy before OTC and those with nonmalignant diseases (other than Turner syndrome).

Our group previously investigated the association between inflammation markers and fertility in men.³¹ In a study performed on male patients with lymphoma facing chemotherapy who cryopreserved sperm, we showed that inflammation markers, such as albumin, hemoglobin, and the presence of B symptoms, are associated with abnormal sperm parameters. Although the negative effect of inflammation on sperm production is well known, the possible effect of tumor-related inflammation on the existing follicular pool and folliculogenesis is understudied.

Interestingly, we found that some of the inflammation markers evaluated—number of lymphocytes and LDH—are independently and positively associated with the number of mature oocytes and IVM rate (LDH). The association between low-grade inflammation and folliculogenesis and the key physiological role of inflammation in folliculogenesis and ovulation were previously demonstrated in several studies,^32,33 mostly involving patients with polycystic ovary syndrome (PCOS).^32–34 These studies suggested that the inflammation process affects follicular dynamics and that in PCOS, hyperinsulinemia, and obesity, inflammation promotes the recruitment of primordial follicles into a cohort of growing follicles.^34,35 The recruitment of early-stage follicles into active follicular development process, even if this follicular dynamics is aberrant, may explain higher numbers and rates of oocytes maturation using IVM in elevated LDH levels, though, of course, it does not reflect the oocytes’ quality and fertilization potential, which might be impaired by this process.

A previous study by Sonigo et al.³⁶ examined the feasibility of IVM for urgent FP in a group of 25 patients with HL and NHL. They could not find an association between lymphomas’ characteristics (stage, B symptoms, and performance) and the number of recovered or frozen oocytes by IVM. Although our findings are in agreement with their results on B symptoms in patients with HL and NHL, we were additionally able to explore the effect of chemotherapy exposure on outcomes of patients with lymphoma and the unique group of patients with leukemia, often requiring urgent FP.

The AML group, though small, showed poorer outcomes in the chance for acquiring immature oocytes and a trend toward lower IVM (though not significant). This can be explained by the fact that all patients in this group received cytotoxic chemotherapy in comparison to ALL and lymphoma groups that included both chemotherapy-naive and exposed patients. However, this explanation does not fully clarify this difference considering that alkylating agents are typically not part of the initial treatment regimen for AML, in contrast to ALL.

A study by Wu et al.³⁷ also showed that in a small group of patients with malignant and nonmalignant conditions, before chemotherapy or BMT, oocyte or embryo freezing is feasible, even in patients previously exposed to chemo. We were able to investigate a larger, similar group of malignant-only conditions and show the potential of immediate FP options regarding IVM outcomes without prior random start controlled ovarian hyperstimulation.

It has been previously implied that following chemotherapy, a recovery period of the remaining follicles in the tissue is required to enable its function later. The “burn-out” phenomenon, described by Gavish et al.,³⁸ claims to result from chemotherapy exposure, especially to alkylating agents. One might speculate that sometime after chemotherapy, some of the oocytes will join the “early-maturing” pool, enabling their collection and IVM in the lab. This may explain our results, showing that although previous chemotherapy exposure is associated with the number of collected immature oocytes in patients with HL, it does not appear to affect the potential to mature them in vitro as demonstrated here in the similar IVM rate.

The safe time interval from chemotherapy exposure to oocyte maturation for future use is unknown in humans and is a major safety issue. Clinical practice suggests avoiding the use of oocytes for at least 3 months following chemotherapy exposure. Previous studies investigating mice models for the feasibility of IVM shortly after exposure to alkylating agents, such as cyclophosphamide, demonstrated that many of the secondary follicles collected from pre-menarche mice after recent cyclophosphamide treatment had normal spindle and chromosome configurations following IVM.³⁹ Nevertheless, human oocytes retrieved shortly after chemotherapy should not be used because of possible DNA damage.

Our study has several limitations. The main limitations are the relatively small population, the long time period of this study, and the different chemotherapy protocols. However, the IVM technique did not change in our center over that time period. Another aspect to consider in our study is that although we provide the count of ampules containing tissue fragments (each containing approximately five ovarian tissue fragments), we could not report the exact total number of tissue fragments per patient. Incorporating these data could enhance the accuracy and depth of our analysis. An additional obvious limitation is the introduction of new drugs with reduced cytotoxic effects for patients with lymphoma and leukemia, hampering the ability to conclude from our data regarding OTC and IVM results, following recent and more updated guidelines and chemotherapy regimen protocols. However, our data contribute to the understanding of the IVM process potential in different hematological malignancies and may be useful for future investigations. We did not refer to stage and specific chemotherapy duration and number of cycles in our analyses because of the small study groups, and the generalizability and clinical significance of the parameters identified in the regression models are constrained by the small sample size. To conclude, OTC and IVM are feasible in both patients with lymphoma and leukemia. The use of IVM, even after exposure to chemotherapy, should be further studied and evaluated for its safety and possible use for FP purposes in these and other malignancies.

Footnotes

Authors’ Contributions

G.K. and E.G.-I.: Conceptualization, methodology, formal analysis, investigation, data curation, visualization, and writing—original draft preparation. K.V.: Formal analysis, investigation, data curation, visualization, and writing—reviewing and editing. A.D. and T.M.-D.: Methodology, investigation, and visualization. R.G.S. and N.C.: Conceptualization, methodology, formal analysis, investigation, data curation, visualization, and writing—original draft preparation, reviewing, and editing. T.I.: Conceptualization, methodology, writing—reviewing and editing, supervision, and project administration.

Data Availability Statement

The datasets used and analyzed during the current study are not publicly available because of containing sensitive personal data regarding patients with infertility but are available from the corresponding author on reasonable request.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this study.

Supplementary Material

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Supplementary Material

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